Compositions and methods for siRNA inhibition of HIF-1 alpha

ABSTRACT

RNA interference using small interfering RNAs which target HIF-1 alpha mRNA inhibit expression of the HIF-1 alpha gene. As HIF-1 alpha is a transcriptional regulator of VEGF, expression of VEGF is also inhibited. Control of VEGF production through siRNA-mediated down-regulation of HIF-1 alpha can be used to inhibit angiogenesis, in particularly in diseases such as diabetic retinopathy, age related macular degeneration and many types of cancer.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional patentapplication serial No. 60/423,262, filed on Nov. 1, 2002.

FIELD OF THE INVENTION

[0002] This invention relates to the regulation of gene expression bysiRNA-induced degradation of the transcriptional regulator HIF-1 alpha.In particular, genes in the VEGF mitogenic pathway can bedown-regulated.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis, defined as the growth of new capillary bloodvessels, plays a fundamental role in growth and development. In maturehumans, the ability to initiate an angiogenic response is present in alltissues, but is held under strict control. A key regulator ofangiogenesis is vascular endothelial growth factor (“VEGF”), also calledvascular permeability factor (“VPF”).

[0004] VEGF is expressed in abnormally high levels in certain tissuesfrom diseases characterized by aberrant angiogenesis, such as cancers,diabetic retinopathy, psoriasis, age-related macular degeneration,rheumatoid arthritis and other inflammatory diseases. Therefore, agentswhich selectively decrease the VEGF levels in these tissues can be usedto treat cancer and other angiogenic diseases.

[0005] Hypoxia-inducible factor 1 (HIF-1) is a heterodimericbasic-helix-loop-helix-PAS transcription factor consisting of HIF-1alpha and HIF-1 beta subunits. HIF-1 alpha expression and HIF-1transcriptional activity increase exponentially as cellular oxygenconcentration is decreased. Several dozen target genes that aretransactivated by HIF-1 have been identified, including those encodingerythropoietin, glucose transporters, glycolytic enzymes, and VEGF.Semenza GL (1999), Ann. Rev. Cell. Dev. Biol. 15: 551-578.

[0006] Loss of p53 in tumor cells enhances HIF-1 alpha levels andaugments HIF-1-dependent transcriptional activation of VEGF in responseto hypoxia. Forced expression of HIF-1 alpha in p53-expressing tumorcells increases hypoxia-induced VEGF expression and augmentsneovascularization and growth of tumor xenografts. These resultsindicate that amplification of normal HIF-1-dependent responses tohypoxia via loss of p53 function contributes to the angiogenic switchduring tumorigenesis. Ravi R. et al. (2000), Genes Dev. 14: 34-44.

[0007] RNA interference (“RNAi”) is a method of post-transcriptionalgene regulation that is conserved throughout many eukaryotic organisms.RNAi is induced by short (i.e., <30 nucleotide) double stranded RNA(“dsRNA”) molecules which are present in the cell (Fire A et al. (1998),Nature 391: 806-811). These short dsRNA molecules, called “shortinterfering RNA” or “siRNA,” cause the destruction of messenger RNAs(“mRNAs”) which share sequence homology with the siRNA to within onenucleotide resolution (Elbashir S M et al. (2001), Genes Dev, 15:188-200). It is believed that the siRNA and the targeted mRNA bind to anRNA-induced silencing complex (“RISC”), which cleaves the targeted mRNA.The siRNA is apparently recycled much like a multiple-turnover enzyme,with 1 siRNA molecule capable of inducing cleavage of approximately 1000mRNA molecules. siRNA-mediated RNAi is therefore more effective thanother currently available technologies for inhibiting expression of atarget gene.

[0008] Elbashir S M et al. (2001), supra, has shown that synthetic siRNAof 21 and 22 nucleotides in length, and which have short 3′ overhangs,can induce RNAi of target mRNA in a Drosophila cell lysate. Culturedmammalian cells also exhibit RNAi with synthetic siRNA (Elbashir S M etal. (2001) Nature, 411: 494-498), and RNAi induced by synthetic siRNAhas recently been shown in living mice (McCaffrey A P et al. (2002),Nature, 418: 38-39; Xia H et al. (2002), Nat. Biotech. 20: 1006-1010).The therapeutic potential of siRNA-mediated RNAi has been demonstratedby several recent in vitro studies, including the siRNA-directedinhibition of HIV-1 infection (Novina C D et al. (2002), Nat. Med. 8:681-686) and reduction of neurotoxic polyglutamine disease proteinexpression (Xia H et al. (2002), supra). Therapeutic RNAi has also beendemonstrated in human cancer cells by Alan Gewirtz, as described inpublished U.S. patent application US 2002/0173478.

[0009] It has now been found that siRNA-induced RNAi of HIF-1 alpharesults in the destruction of HIF-1 alpha mRNA, with a concomitantreduction in VEGF expression and inhibition of angiogenesis.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to siRNAs which specificallytarget and cause RNAi-induced degradation of mRNA from the human HIF-1alpha gene. The siRNA compounds and compositions of the invention areused to treat cancerous tumors and other angiogenic diseases andnon-pathogenic conditions in which VEGF is overexpressed in tissues inor near the area of neovascularization.

[0011] Thus, the invention provides siRNA, and pharmaceuticalcompositions thereof, which target HIF-1 alpha mRNA and induceRNAi-mediated degradation of the targeted mRNA.

[0012] The invention further provides a method of inhibiting expressionof HIF-1 alpha, comprising administering to a subject an effectiveamount of an siRNA targeted to HIF-1 alpha mRNA, such that the HIF-1alpha mRNA is degraded.

[0013] The invention further provides a method of inhibitingangiogenesis, comprising administering an effective amount of an siRNAtargeted to HIF-1 alpha mRNA to a subject, such that the HIF-1 alphamRNA is degraded and the expression of VEGF is inhibited.

[0014] The invention further provides a method of treating an angiogenicdisease, comprising administering an effective amount of an siRNAtargeted to HIF-1 alpha mRNA to a subject, such that the HIF-1 alphamRNA is degraded and the expression of VEGF is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a histogram of VEGF concentration, as measured by VEGFELISA at OD₄₅₀ nanometers, in non-hypoxic (“−”) cultured HEK-293 cellstreated with no siRNA (“no”), and in hypoxic (“+”) cultured HEK-293cells treated with: no siRNA (“no”); nonspecific siRNA (“EGFP”); or withtwenty separate siRNAs targeting human HIF-1 alpha mRNA (“hHIF1#1-20”).

[0016]FIG. 2 is a histogram showing cytotoxicity in non-hypoxic (“−”)cultured HEK-293 cells treated with no siRNA (“no”), and in hypoxic(“+”) cultured HEK-293 cells treated with: no siRNA (“no”); nonspecificsiRNA (“EGFP”); or with twenty separate siRNAs targeting human HIF-1alpha mRNA (“hHIF1#1-20”).

[0017]FIG. 3 is a histogram showing the area of choroidalneovascularization in mm², in eyes from control mice (“control”) andmice treated with anti-HIF-1 alpha siRNA (“HIF-1 siRNA”).

DETAILED DESCRIPTION OF THE INVENTION

[0018] Compositions and methods comprising siRNA targeted to HIF-1 alphamRNA are advantageously used to inhibit angiogenesis, in particular forthe treatment of angiogenic diseases. The siRNA of the invention causesRNAi-mediated destruction of the HIF-1 alpha mRNA. HIF-1 alpha is atranscriptional regulator of VEGF, and the reduction in HIF-1 alpha mRNAcaused by the siRNA of the invention is correlated with a reduction inVEGF production. Because VEGF is required for initiating and maintainingangiogenesis, the siRNA-mediated destruction of HIF-1 alpha slows, stopsor reverses the angiogenic process.

[0019] As used herein, siRNA which is “targeted to the HIF-1 alpha mRNA”means siRNA in which a first strand of the duplex has the samenucleotide sequence as a portion of the HIF-1 mRNA sequence. It isunderstood that the second strand of the siRNA duplex is complementaryto both the first strand of the siRNA duplex and to the same portion ofthe HIF-1 alpha mRNA.

[0020] The invention therefore provides isolated siRNA comprising shortdouble-stranded RNA from about 17 nucleotides to about 29 nucleotides inlength, preferably from about 19 to about 25 nucleotides in length, thatare targeted to the target mRNA. The siRNA comprise a sense RNA strandand a complementary antisense RNA strand annealed together by standardWatson-Crick base-pairing interactions (hereinafter “base-paired”). Asis described in more detail below, the sense strand comprises a nucleicacid sequence which is substantially identical to a target sequencecontained within the target mRNA.

[0021] As used herein, a nucleic acid sequence “substantially identical”to a target sequence contained within the target mRNA is a nucleic acidsequence which is identical to the target sequence, or which differsfrom the target sequence by one or more nucleotides. Sense strands ofthe invention which comprise nucleic acid sequences substantiallyidentical to a target sequence are characterized in that siRNAcomprising such sense strands induce RNAi-mediated degradation of mRNAcontaining the target sequence. For example, an siRNA of the inventioncan comprise a sense strand comprise nucleic acid sequences which differfrom a target sequence by one, two or three or more nucleotides, as longas RNAi-mediated degradation of the target mRNA is induced by the siRNA.

[0022] The sense and antisense strands of the present siRNA can comprisetwo complementary, single-stranded RNA molecules or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area. Withoutwishing to be bound by any theory, it is believed that the hairpin areaof the latter type of siRNA molecule is cleaved intracellularly by the“Dicer” protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra). As describedbelow, the siRNA can also contain alterations, substitutions ormodifications of one or more ribonucleotide bases. For example, thepresent siRNA can be altered, substituted or modified to contain one ormore deoxyribonucleotide bases.

[0023] As used herein, “isolated” means synthetic, or altered or removedfrom the natural state through human intervention. For example, an siRNAnaturally present in a living animal is not “isolated,” but a syntheticsiRNA, or an siRNA partially or completely separated from the coexistingmaterials of its natural state is “isolated.” An isolated siRNA canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a cell into which the siRNA has beendelivered.

[0024] As used herein, “target mRNA” means human HIF-1 alpha mRNA,mutant or alternative splice forms of human HIF-1 alpha mRNA, or mRNAfrom cognate HIF-1 alpha genes. A cDNA sequence corresponding to a humanHIF-1 alpha mRNA sequence is given in SEQ ID NO: 1.

[0025] Splice variants of human HIF-1 alpha are known, including HIF-1alpha transcript variants 1 (SEQ ID NO: 2) and 2 (SEQ ID NO: 3), asdescribed in GenBank record accession nos. NM_(—)001530 andNM_(—)181054, the entire disclosures of which are herein incorporated byreference. The mRNA transcribed from the human HIF-1 alpha gene can beanalyzed for further alternative splice forms using techniqueswell-known in the art. Such techniques include reversetranscription-polymerase chain reaction (RT-PCR), northern blotting andin-situ hybridization. Techniques for analyzing mRNA sequences aredescribed, for example, in Busting S A (2000), J. Mol. Endocrinol. 25:169-193, the entire disclosure of which is herein incorporated byreference. Representative techniques for identifying alternativelyspliced mRNAs are also described below.

[0026] For example, databases that contain nucleotide sequences relatedto a given disease gene can be used to identify alternatively splicedmRNA. Such databases include GenBank, Embase, and the Cancer GenomeAnatomy Project (CGAP) database. The CGAP database, for example,contains expressed sequence tags (ESTs) from various types of humancancers. An mRNA or gene sequence from the HIF-1 alpha gene can be usedto query such a database to determine whether ESTs representingalternatively spliced mRNAs have been found for a these genes.

[0027] A technique called “RNAse protection” can also be used toidentify alternatively spliced HIF-1 alpha mRNA. RNAse protectioninvolves translation of a gene sequence into synthetic RNA, which ishybridized to RNA derived from other cells; for example, cells fromtissue at or near the site of neovascularization. The hybridized RNA isthen incubated with enzymes that recognize RNA:RNA hybrid mismatches.Smaller than expected fragments indicate the presence of alternativelyspliced mRNAs. The putative alternatively spliced mRNAs can be clonedand sequenced by methods well known to those skilled in the art.

[0028] RT-PCR can also be used to identify alternatively spliced HIF-1alpha mRNA. In RT-PCR, mRNA from a tissue is converted into cDNA by theenzyme reverse transcriptase, using methods well-known to those ofordinary skill in the art. The entire coding sequence of the cDNA isthen amplified via PCR using a forward primer located in the 3′untranslated region, and a reverse primer located in the 5′ untranslatedregion. The amplified products can be analyzed for alternative spliceforms, for example by comparing the size of the amplified products withthe size of the expected product from normally spliced mRNA, e.g., byagarose gel electrophoresis. Any change in the size of the amplifiedproduct can indicate alternative splicing.

[0029] The mRNA produced from a mutant HIF-1 alpha gene can also bereadily identified through the techniques described above foridentifying alternative splice forms. As used herein, “mutant” HIF-1alpha gene or mRNA includes a HIF-1 alpha gene or mRNA which differs insequence from the HIF-1 alpha mRNA sequences set forth herein. Thus,allelic forms of HIF-1 alpha genes, and the mRNA produced from them, areconsidered “mutants” for purposes of this invention.

[0030] As used herein, a gene or mRNA which is “cognate” to human HIF-1alpha is a gene or mRNA from another mammalian species which ishomologous to human HIF-1 alpha. For example, the cognate HIF-1 alphamRNA from the rat and mouse are described in GenBank record accessionnos. NM_(—)024359 and NM_(—)010431, respectively, the entire disclosureof which is herein incorporated by reference. The rat HIF-1 alpha mRNAsequence is given in SEQ ID NO: 4, and the mouse HIF-1 alpha mRNAsequence is given in SEQ ID NO: 5.

[0031] It is understood that human HIF-1 alpha mRNA may contain targetsequences in common with their respective alternative splice forms,cognates or mutants. A single siRNA comprising such a common targetingsequence can therefore induce RNAi-mediated degradation of different RNAtypes which contain the common targeting sequence.

[0032] The siRNA of the invention can comprise partially purified RNA,substantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, or modifications that make the siRNA resistantto nuclease digestion, or the substitution of one or more nucleotides inthe siRNA with deoxyribonucleotides.

[0033] One or both strands of the siRNA of the invention can alsocomprise a 3′ overhang. As used herein, a “3′ overhang” refers to atleast one unpaired nucleotide extending from the 3′-end of a duplexedRNA strand.

[0034] Thus in one embodiment, the siRNA of the invention comprises atleast one 3′ overhang of from 1 to about 6 nucleotides (which includesribonucleotides or deoxyribonucleotides) in length, preferably from 1 toabout 5 nucleotides in length, more preferably from 1 to about 4nucleotides in length, and particularly preferably from about 2 to about4 nucleotides in length.

[0035] In the embodiment in which both strands of the siRNA moleculecomprise a 3′ overhang, the length of the overhangs can be the same ordifferent for each strand. In a most preferred embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA of the invention cancomprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid(“uu”).

[0036] In order to enhance the stability of the present siRNA, the 3′overhangs can be also stabilized against degradation. In one embodiment,the overhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

[0037] In certain embodiments, the siRNA of the invention comprises thesequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNAcomprise approximately 30-70% G/C, and preferably comprise approximately50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TTor N21 (i.e., positions 3 to 23), respectively. In the latter case, the3′ end of the sense siRNA is converted to TT. The rationale for thissequence conversion is to generate a symmetric duplex with respect tothe sequence composition of the sense and antisense strand 3′ overhangs.The antisense strand is then synthesized as the complement to positions1 to 21 of the sense strand.

[0038] Because position 1 of the 23-nt sense strand in these embodimentsis not recognized in a sequence-specific manner by the antisense strand,the 3′-most nucleotide residue of the antisense strand can be chosendeliberately. However, the penultimate nucleotide of the antisensestrand (complementary to position 2 of the 23-nt sense strand in eitherembodiment) is generally complementary to the targeted sequence.

[0039] In another embodiment, the siRNA of the invention comprises thesequence NAR(N17)YNN, where R is a purine (e.g., A or G) and Y is apyrimidine (e.g., C or U/T). The respective 21-nt sense and antisensestrands of this embodiment therefore generally begin with a purinenucleotide. Such siRNA can be expressed from pol III expression vectorswithout a change in targeting site, as expression of RNAs from pol IIIpromoters is only believed to be efficient when the first transcribednucleotide is a purine.

[0040] The siRNA of the invention can be targeted to any stretch ofapproximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA are given, for example, in Tuschl T et al., “ThesiRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Department of Cellular Biochemistry, AG 105, Max-Planck-Institute forBiophysical Chemistry, 37077 Göttingen, Germany, and can be found byaccessing the website of the Max Planck Institute and searching with thekeyword “siRNA.” Thus, the sense strand of the present siRNA comprises anucleotide sequence identical to any contiguous stretch of about 19 toabout 25 nucleotides in the target mRNA.

[0041] Generally, a target sequence on the target mRNA can be selectedfrom a given cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon. Asuitable target sequence in the HIF-1 alpha cDNA sequence is:AACTGGACACAGTGTGTTTGA SEQ ID NO: 6

[0042] Thus, an siRNA of the invention targeting this sequence, andwhich has 3′ UU overhangs (overhangs shown in bold) is:      5′-aacuaacuggacacagugugu uu-3′ SEQ ID NO: 7 3′-uuuugauugaccugugucacaca-5′ SEQ ID NO: 8

[0043] An siRNA of the invention targeting this same sequence, buthaving 3′ TT overhangs on each strand (overhangs shown in bold) is:   5′-aacuaacuggacacaguguguTT-3′ (SEQ ID NO: 9)3′-TTuugauugaccugugucacaca-5′ (SEQ ID NO: 10)

[0044] Exemplary HIF-1 alpha target sequences from which siRNA of theinvention can be derived include those in Table 1 and those given in SEQID NOS: 39-298. TABLE 1 HIF-1 Alpha Target Sequences target sequence SEQID NO: AACTAACTGGACACAGTGTGT 11 CGACAAGAAAAAGATAA 12 AAAGATAAGTTCTGAAC13 AGATAAGTTCTGAACGT 14 GTTCTGAACGTCGAAAA 15 AAGAAAAGTCTCGAGAT 16GAAAAGTCTCGAGATGC 17 AGTCTCGAGATGCAGCC 18 GTAAAGAATCTGAAGTT 19GAATCTGAAGTTTTTTA 20 GTTTTTTATGAGCTTGC 21 GGCCTCTGTGATGAGGC 22CTTCTGGATGCTGGTGA 23 AGCACAGATGAATTGCT 24 AAATGCTTACACACAGAAATG 25GAAAAAGATAAGTTCTG 26 AAGATAAGTTCTGAACG 27 GATAAGTTCTGAACGTC 28CGTCGAAAAGAAAAGTC 29 AGAAAAGTCTCGAGATG 30 AAGTCTCGAGATGCAGC 31GTCTCGAGATGCAGCCA 32 AGAATCTGAAGTTTTTT 33 TCTGAAGTTTTTTATGA 34TGTGAGTTCGCATCTTG 35 ACTTCTGGATGCTGGTG 36 GATGACATGAAAGCACA 37GCACAGATGAATTGCTT 38

[0045] The siRNA of the invention can be obtained using a number oftechniques known to those of skill in the art. For example, the siRNAcan be chemically synthesized or recombinantly produced using methodsknown in the art, such as the Drosophila in vitro system described inU.S. published application 2002/0086356 of Tuschl et al., the entiredisclosure of which is herein incorporated by reference.

[0046] Preferably, the siRNA of the invention are chemically synthesizedusing appropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

[0047] Alternatively, siRNA can also be expressed from recombinantcircular or linear DNA plasmids using. any suitable promoter. Suitablepromoters for expressing siRNA of the invention from a plasmid include,for example, the U6 or H1 RNA pol III promoter sequences and thecytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant plasmids of the inventioncan also comprise inducible or regulatable promoters for expression ofthe siRNA in a particular tissue or in a particular intracellularenvironment.

[0048] The siRNA expressed from recombinant plasmids can either beisolated from cultured cell expression systems by standard techniques,or can be expressed intracellularly at or near the area ofneovascularization in vivo. The use of recombinant plasmids to deliversiRNA of the invention to cells in vivo is discussed in more detailbelow.

[0049] The siRNA of the invention can be expressed from a recombinantplasmid either as two separate, complementary RNA molecules, or as asingle RNA molecule with two complementary regions.

[0050] Selection of plasmids suitable for expressing siRNA of theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the plasmid, and methods of delivering the recombinantplasmid to the cells of interest are within the skill in the art. See,for example Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448; BrummelkampT R et al. (2002), Science 296: 550-553; Miyagishi M et al. (2002), Nat.Biotechnol. 20: 497-500; Paddison P J et al. (2002), Genes Dev. 16:948-958; Lee N S et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul CP et al. (2002), Nat. Biotechnol. 20: 505-508, the entire disclosures ofwhich are herein incorporated by reference.

[0051] For example, a plasmid can comprise a sense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter, and an antisense RNA strandcoding sequence in operable connection with a polyT termination sequenceunder the control of a human U6 RNA promoter.

[0052] As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

[0053] As used herein, “under the control” of a promoter means that thenucleic acid sequences encoding the sense or antisense strands arelocated 3′ of the promoter, so that the promoter can initiatetranscription of the sense or antisense coding sequences.

[0054] The siRNA of the invention can also be expressed from recombinantviral vectors intracellularly at or near the area of neovascularizationin vivo. The recombinant viral vectors of the invention comprisesequences encoding the siRNA of the invention and any suitable promoterfor expressing the siRNA sequences. Suitable promoters include, forexample, the U6 or H1 RNA pol III promoter sequences and thecytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the siRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver siRNA of the invention to cells in vivo is discussed in moredetail below.

[0055] The siRNA of the invention can be expressed from a recombinantviral vector either as two separate, complementary nucleic acidmolecules, or as a single nucleic acid molecule with two complementaryregions.

[0056] Any viral vector capable of accepting the coding sequences forthe siRNA molecule(s) to be expressed can be used, for example vectorsderived from adenovirus (AV); adeno-associated virus (AAV); retroviruses(e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpesvirus, and the like. The tropism of the viral vectors can also bemodified by pseudotyping the vectors with envelope proteins or othersurface antigens from other viruses. For example, an AAV vector of theinvention can be pseudotyped with surface proteins from vesicularstomatitis virus (VSV), rabies, Ebola, Mokola, and the like.

[0057] Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Domburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;and Anderson W F (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

[0058] Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

[0059] A suitable AV vector for expressing the siRNA of the invention, amethod for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

[0060] Suitable AAV vectors for expressing the siRNA of the invention,methods for constructing the recombinant AAV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J.Virol., 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826;U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

[0061] The ability of an siRNA containing a given target sequence tocause RNAi-mediated degradation of the target mRNA can be evaluatedusing standard techniques for measuring the levels of RNA or protein incells. For example, siRNA of the invention can be delivered to culturedcells, and the levels of target mRNA can be measured by Northern blot ordot blotting techniques, or by quantitative RT-PCR. Alternatively, thelevels of HIF-1 alpha protein in the cultured cells can be measured byELISA or Western blot. A suitable cell culture system for measuring theeffect of the present siRNA on target mRNA or protein levels isdescribed in Example 1 below.

[0062] The ability of an siRNA to target and cause RNAi-mediateddegradation of HIF-1 alpha mRNA can also be evaluated by measuring thelevels of VEGF mRNA or protein in cultured cells, as a reduction inHIF-1 alpha expression will also inhibit VEGF expression.

[0063] For example, 50% confluent 293 human kidney cells can beincubated with culture medium containing an siRNA (optionally complexedto a transfection reagent such as Mirus Transit TKO transfectionreagent) for 48 hours, followed by ELISA or mRNA quantification ofeither HIF-1 alpha or VEGF. Cells incubated with an siRNA not homologousto the HIF-1 alpha target sequence can be used as controls.

[0064] RNAi-mediated degradation of target mRNA by an siRNA containing agiven target sequence can also be evaluated with animal models ofneovascularization, such as the retinopathy of prematurity (“ROP”) orchoroidal neovascularization (“CNV”) mouse models. For example, areas ofneovascularization in an ROP or CNV mouse can be measured before andafter administration of an siRNA. A reduction in the areas ofneovascularization in these models upon administration of the siRNAindicates the down-regulation of the target mRNA (see Example 2 below).

[0065] As discussed above, the siRNA of the invention target and causethe RNAi-mediated degradation of HIF-1 alpha mRNA, or alternative spliceforms, mutants or cognates thereof. Degradation of the target mRNA bythe present siRNA reduces the production of a functional gene productfrom the HIF-1 alpha gene. Thus, the invention provides a method ofinhibiting expression of HIF-1 alpha in a subject, comprisingadministering an effective amount of an siRNA of the invention to thesubject, such that the target mRNA is degraded. In the practice of thepresent methods, it is understood that more than one siRNA of theinvention can be administered simultaneously to the subject.

[0066] Without wishing to be bound by any theory, the products of theHIF-1 alpha gene are believed to be involved in the transcriptionalregulation of VEGF. VEGF is in turn required for initiating andmaintaining angiogenesis. Thus, the invention also provides a method ofinhibiting angiogenesis in a subject by the RNAi-mediated degradation ofthe target mRNA by an siRNA of the invention.

[0067] As used herein, a “subject” includes a human being or non-humananimal. Preferably, the subject is a human being.

[0068] As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA, or anamount sufficient to inhibit angiogenesis in a subject.

[0069] RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

[0070] Inhibition of angiogenesis can be evaluated by directly measuringthe progress of pathogenic or nonpathogenic angiogenesis in a subject;for example, by observing the size of a neovascularized area before andafter treatment with the siRNA of the invention. An inhibition ofangiogenesis is indicated if the size of the neovascularized area staysthe same or is reduced. Techniques for observing and measuring the sizeof neovascularized areas in a subject are within the skill in the art;for example, areas of choroid neovascularization can be observed byophthalmoscopy.

[0071] Inhibition of angiogenesis can also be inferred through observinga change or reversal in a pathogenic condition associated with theangiogenesis. For example, in ARMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site. Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA of the invention.

[0072] It is understood that the siRNA of the invention can degrade thetarget mRNA (and thus inhibit angiogenesis) in substoichiometricamounts. Without wishing to be bound by any theory, it is believed thatthe siRNA of the invention induces the RISC to degrade of the targetmRNA in a catalytic manner. Thus, compared to standard anti-angiogenictherapies, significantly less siRNA needs to be delivered at or near thesite of neovascularization to have a therapeutic effect.

[0073] One skilled in the art can readily determine an effective amountof the siRNA of the invention to be administered to a given subject, bytaking into account factors such as the size and weight of the subject;the extent of the neovascularization or disease penetration; the age,health and sex of the subject; the route of administration; and whetherthe administration is regional or systemic. Generally, an effectiveamount of the siRNA of the invention comprises an amount which providesan intercellular concentration at or near the neovascularization site offrom about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nMto about 50 nM, more preferably from about 2.5 nM to about 10 nM. It iscontemplated that greater or lesser amounts of siRNA can beadministered.

[0074] The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendometrial neovascularization, and processes involved in the productionof fatty tissues or cholesterol. Thus, the invention provides a methodfor inhibiting non-pathogenic angiogenesis, e.g., for controlling weightor promoting fat loss, for reducing cholesterol levels, or as anabortifacient.

[0075] The present methods can also inhibit angiogenesis which isassociated with an angiogenic disease; i.e., a disease in whichpathogenicity is associated with inappropriate or uncontrolledangiogenesis. For example, most cancerous solid tumors generate anadequate blood supply for themselves by inducing angiogenesis in andaround the tumor site. This tumor-induced angiogenesis is often requiredfor tumor growth, and also allows metastatic cells to enter thebloodstream.

[0076] Other angiogenic diseases include diabetic retinopathy,age-related macular degeneration (ARMD), psoriasis, rheumatoid arthritisand other inflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in ARMD, the choroid is invaded anddestroyed by capillaries. The angiogenesis-driven destruction of thechoroid in ARMD eventually leads to partial or full blindness.

[0077] Preferably, an siRNA of the invention is used to inhibit thegrowth or metastasis of solid tumors associated with cancers; forexample breast cancer, lung cancer, head and neck cancer, brain cancer,abdominal cancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma; skin cancer(e.g., melanoma), lymphomas and blood cancer.

[0078] More preferably, an siRNA of the invention is used to inhibitchoroidal neovascularization in age-related macular degeneration.

[0079] For treating angiogenic diseases, the siRNA of the invention canadministered to a subject in combination with a pharmaceutical agentwhich is different from the present siRNA. Alternatively, the siRNA ofthe invention can be administered to a subject in combination withanother therapeutic method designed to treat the angiogenic disease. Forexample, the siRNA of the invention can be administered in combinationwith therapeutic methods currently employed for treating cancer orpreventing tumor metastasis (e.g., radiation therapy, chemotherapy, andsurgery). For treating tumors, the siRNA of the invention is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

[0080] In the present methods, the present siRNA can be administered tothe subject either as naked siRNA, in conjunction with a deliveryreagent, or as a recombinant plasmid or viral vector which expresses thesiRNA.

[0081] Suitable delivery reagents for administration in conjunction withthe present siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

[0082] Liposomes can aid in the delivery of the siRNA to a particulartissue, such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

[0083] Preferably, the liposomes encapsulating the present siRNAcomprise a ligand molecule that can target the liposome to a particularcell or tissue at or near the site of angiogenesis. Ligands which bindto receptors prevalent in tumor or vascular endothelial cells, such asmonoclonal antibodies that bind to tumor antigens or endothelial cellsurface antigens, are preferred.

[0084] Particularly preferably, the liposomes encapsulating the presentsiRNA are modified so as to avoid clearance by the mononuclearmacrophage and reticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

[0085] Opsonization-inhibiting moieties for use in preparing theliposomes of the invention are typically large hydrophilic polymers thatare bound to the liposome membrane. As used herein, an opsonizationinhibiting moiety is “bound” to a liposome membrane when it ischemically or physically attached to the membrane, e.g., by theintercalation of a lipid-soluble anchor into the membrane itself, or bybinding directly to active groups of membrane lipids. Theseopsonization-inhibiting hydrophilic polymers form a protective surfacelayer which significantly decreases the uptake of the liposomes by themacrophage-monocyte system (“MMS”) and reticuloendothelial system(“RES”); e.g., as described in U.S. Pat. No. 4,920,016, the entiredisclosure of which is herein incorporated by reference. Liposomesmodified with opsonization-inhibition moieties thus remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes.

[0086] Stealth liposomes are known to accumulate in tissues fed byporous or “leaky” microvasculature. Thus, target tissue characterized bysuch microvasculature defects, for example solid tumors, willefficiently accumulate these liposomes; see Gabizon, et al. (1988),P.N.A.S., USA, 18: 6949-53. In addition, the reduced uptake by the RESlowers the toxicity of stealth liposomes by preventing significantaccumulation in the liver and spleen. Thus, liposomes of the inventionthat are modified with opsonization-inhibition moieties can deliver thepresent siRNA to tumor cells.

[0087] Opsonization inhibiting moieties suitable for modifying liposomesare preferably water-soluble polymers with a number-average molecularweight from about 500 to about 40,000 daltons, and more preferably fromabout 2,000 to about 20,000 daltons. Such polymers include polyethyleneglycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxyPEG or PPG, and PEG or PPG stearate; synthetic polymers such aspolyacrylamide or poly N-vinyl pyrrolidone; linear, branched, ordendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g.,polyvinylalcohol and polyxylitol to which carboxylic or amino groups arechemically linked, as well as gangliosides, such as ganglioside GM₁.Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof,are also suitable. In addition, the opsonization inhibiting polymer canbe a block copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

[0088] Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

[0089] The opsonization inhibiting moiety can be bound to the liposomemembrane by any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

[0090] Recombinant plasmids which express siRNA of the invention arediscussed above. Such recombinant plasmids can also be administered to asubject directly or in conjunction with a suitable delivery reagent,including the Mirus Transit LT1 lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.Recombinant viral vectors which express siRNA of the invention are alsodiscussed above, and methods for delivering such vectors to an area ofneovascularization in a subject are within the skill in the art.

[0091] The siRNA of the invention can be administered to the subject byany means suitable for delivering the siRNA to the cells of the tissueat or near the area of neovascularization. For example, the siRNA can beadministered by gene gun, electroporation, or by other suitableparenteral or enteral administration routes.

[0092] Suitable enteral administration routes include oral, rectal, orintranasal delivery.

[0093] Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration (e.g., peri-tumoral and intra-tumoral injection,intra-retinal injection or subretinal injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); direct (e.g., topical) application to the area at or near thesite of neovascularization, for example by a catheter or other placementdevice (e.g., a corneal pellet or a suppository, eye-dropper, or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Suitable placement devices include the ocular implantsdescribed in U.S. Pat. Nos. 5,902,598 and 6,375,972, and thebiodegradable ocular implants described in U.S. Pat. No 6,331,313, theentire disclosures of which are herein incorporated by reference. Suchocular implants are available from Control Delivery Systems, Inc.(Watertown, Mass.) and Oculex Pharmaceuticals, Inc. (Sunnyvale, Calif.).

[0094] In a preferred embodiment, injections or infusions of the siRNAare given at or near the site of neovascularization. For example, thesiRNA of the invention can be delivered to retinal pigment epithelialcells in the eye. Preferably, the siRNA is administered topically to theeye, e.g. in liquid or gel form to the lower eye lid or conjunctivalcul-de-sac, as is within the skill in the art (see, e.g., Acheampong AAet al, 2002, Drug Metabol. and Disposition 30: 421-429, the entiredisclosure of which is herein incorporated by reference).

[0095] Typically, the siRNA of the invention is administered topicallyto the eye in volumes of from about 5 microliters to about 75microliters, for example from about 7 microliters to about 50microliters, preferably from about 10 microliters to about 30microliters. The siRNA of the invention is highly soluble in aqueoussolutions, It is understood that topical instillation in the eye ofsiRNA in volumes greater than 75 microliters can result in loss of siRNAfrom the eye through spillage and drainage. Thus, it is preferable toadminister a high concentration of siRNA (e.g., 100-1000 nM) by topicalinstillation to the eye in volumes of from about 5 microliters to about75 microliters.

[0096] A particularly preferred parenteral administration route isintraocular administration. It is understood that intraocularadministration of the present siRNA can be accomplished by injection ordirect (e.g., topical) administration to the eye, as long as theadministration route allows the siRNA to enter the eye. In addition tothe topical routes of administration to the eye described above,suitable intraocular routes of administration include intravitreal,intraretinal, subretinal, subtenon, peri- and retro-orbital,trans-corneal and trans-scleral administration. Such intraocularadministration routes are within the skill in the art; see, e.g., andAcheampong AA et al, 2002, supra; and Bennett et al. (1996), Hum. GeneTher. 7: 1763-1769 and Ambati J et al., 2002, Progress in Retinal andEye Res. 21: 145-151, the entire disclosures of which are hereinincorporated by reference.

[0097] The siRNA of the invention can be administered in a single doseor in multiple doses. Where the administration of the siRNA of theinvention is by infusion, the infusion can be a single sustained dose orcan be delivered by multiple infusions. Injection of the siRNA directlyinto the tissue is at or near the site of neovascularization preferred.Multiple injections of the siRNA into the tissue at or near the site ofneovascularization are particularly preferred.

[0098] One skilled in the art can also readily determine an appropriatedosage regimen for administering the siRNA of the invention to a givensubject. For example, the siRNA can be administered to the subject once,such as by a single injection or deposition at or near theneovascularization site. Alternatively, the siRNA can be administered toa subject multiple times daily or weekly. For example, the siRNA can beadministered to a subject once weekly for a period of from about threeto about twenty-eight weeks, more preferably from about seven to aboutten weeks. In a preferred dosage regimen, the siRNA is injected at ornear the site of neovascularization (e.g., intravitreally) once a weekfor seven weeks. It is understood that periodic administrations of thesiRNA of the invention for an indefinite length of time may be necessaryfor subjects suffering from a chronic neovascularization disease, suchas wet ARMD or diabetic retinopathy.

[0099] Where a dosage regimen comprises multiple administrations, it isunderstood that the effective amount of siRNA administered to thesubject can comprise the total amount of siRNA administered over theentire dosage regimen.

[0100] The siRNA of the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. As used herein, “pharmaceutical formulations” includeformulations for human and veterinary use. Methods for preparingpharmaceutical compositions of the invention are within the skill in theart, for example as described in Remington's Pharmaceutical Science,17th ed., Mack Publishing Company, Easton, Pa. (1985), the entiredisclosure of which is herein incorporated by reference.

[0101] The present pharmaceutical formulations comprise an siRNA of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, saline solutions (e.g., normal saline or balanced salinesolutions such as Hank's or Earle's balanced salt solutions), 0.4%saline, 0.3% glycine, hyaluronic acid and the like.

[0102] Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

[0103] For topical administration to the eye, conventional intraoculardelivery reagents can be used. For example, pharmaceutical compositionsof the invention for topical intraocular delivery can comprise salinesolutions as described above, corneal penetration enhancers, insolubleparticles, petrolatum or other gel-based ointments, polymers whichundergo a viscosity increase upon instillation in the eye, ormucoadhesive polymers. Preferably, the intraocular delivery reagentincreases comeal penetration, or prolongs preocular retention of thesiRNA through viscosity effects or by establishing physicochemicalinteractions with the mucin layer covering the corneal epithelium.

[0104] Suitable insoluble particles for topical intraocular deliveryinclude the calcium phosphate particles described in U.S. Pat. No.6,355,271 of Bell et al., the entire disclosure of which is hereinincorporated by reference. Suitable polymers which undergo a viscosityincrease upon instillation in the eye includepolyethylenepolyoxypropylene block copolymers such as poloxamer 407(e.g., at a concentration of 25%), cellulose acetophthalate (e.g., at aconcentration of 30%), or a low-acetyl gellan gum such as Gelrite®(available from CP Kelco, Wilmington, Del.). Suitable mucoadhesivepolymers include hydrocolloids with multiple hydrophilic functionalgroups such as carboxyl, hydroxyl, amide and/or sulfate groups; forexample, hydroxypropylcellulose, polyacrylic acid, high-molecular weightpolyethylene glycols (e.g., >200,000 number average molecular weight),dextrans, hyaluronic acid, polygalacturonic acid, and xylocan. Suitablecorneal penetration enhancers include cyclodextrins, benzalkoniumchloride, polyoxyethylene glycol lauryl ether (e.g., Brij® 35),polyoxyethylene glycol stearyl ether (e.g., Brij® 78), polyoxyethyleneglycol oleyl ether (e.g., Brij® 98), ethylene diamine tetraacetic acid(EDTA), digitonin, sodium taurocholate, saponins and polyoxyethylatedcastor oil such as Cremaphor EL.

[0105] For solid compositions, conventional nontoxic solid carriers canbe used; for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like.

[0106] For example, a solid pharmaceutical composition for oraladministration can comprise any of the carriers and excipients listedabove and 10-95%, preferably 25%-75%, of one or more siRNA of theinvention. A pharmaceutical composition for aerosol (inhalational)administration can comprise 0.01-20% by weight, preferably 1%-10% byweight, of one or more siRNA of the invention encapsulated in a liposomeas described above, and propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

[0107] The invention will now be illustrated with the followingnon-limiting examples. The animal experiments described below wereperformed using the University of Pennsylvania institutional guidelinesfor the care and use of animals in research.

EXAMPLE 1 Inhibition of Human VEGF Expression in Cultured HumanEmbryonic Kidney Cells with Anti-HIF-1 Alpha siRNAs

[0108] Human embryonic kidney 293 (HEK-293) cells were cultured in 24well plates at 37° C. with 5% CO₂ overnight, in standard growth medium.Transfections were performed the following day on experimental andcontrol cells, when the cells were approximately 50% confluent. Theexperimental cells were transfected with 25 nM human HIF-1 alpha siRNAmixed in calcium phosphate reagent. Control cells were treated withtransfection reagent lacking siRNA, or with 25 nM nonspecific siRNA(EGFP1 siRNA) in calcium phosphate transfection reagent. For theexperimental cells, twenty different siRNAs targeted to human HIF-1alpha mRNA were tested. These anti-HIF-1 alpha siRNAs contained thetargeting sequences listed in Table 2, and all siRNAs contained 3′ TToverhangs on each strand. The “sample #” listed in Table 2 correspondsto the experimental cell sample as indicated in FIGS. 1 and 2. TABLE 2Target Sequences for Anti-HIF-1 Alpha siRNAs Tested in HEK-293 CellsTarget Sequence SEQ ID NO: Sample # AACTAGCCGAGGAAGAACTAT  76  1AACTGTCATATATAACACCAA 117  2 AATTACGTTGTGAGTGGTATT 122  3AAACGCCAAAGCCACTTCGAA 161  4 AAAGTTCACCTGAGCCTAATA 177  5AAGTTCACCTGAGCCTAATAG 180  6 AAAGCACAGTTACAGTATTCC 200  7AAGCACAGTTACAGTATTCCA 201  8 AAAAGACCGTATGGAAGACAT 212  9AACTACTAGTGCCACATCATC 222 10 AAAGTCGGACAGCCTCACCAA 223 11AAGTCGGACAGCCTCACCAAA 224 12 AACGTGTTATCTGTCGCTTTG 237 13AAGCAGTAGGAATTGGAACAT 255 14 AATGGATGAAAGTGGATTACC 274 15AATGTGAGTTCGCATCTTGAT  40 16 AAGATGACATGAAAGCACAGA  44 17AACTGGACACAGTGTGTTTGA  56 18 AAATTCCTTTAGATAGCAAGA  93 19AAACCGGTTGAATCTTCAGAT 127 20

[0109] At four hours post-transfection, hypoxia was induced in controland experimental HEK-293 cells with desferrioxamine at a finalconcentration of 200 micromolar. At 48 hours post transfection, the cellculture medium was removed from all wells and a human VEGF ELISA (R & Dsystems, Minneapolis, Minn.) was performed as described in theQuantikine human VEGF ELISA protocol. ELISA results were read on anAD340 plate reader (Beckman Coulter), and are given in FIG. 1.

[0110] As can be seen from FIG. 1, human VEGF protein was upregulated inHEK-293 cells by the desferrioxamine-mediated induction of hypoxia. Thehypoxia-induced increase in VEGF protein was reduced in cellstransfected with the human anti-HIF-1 alpha siRNAs. Transfections ofhypoxic cells with non-specific siRNA (EGFP siRNA) or mock transfectionwithout siRNA had no effect on VEGF protein levels. The anti-HIF-1 alphasiRNAs hHIF1#12, hHIF1#13 and hHIF1#16 reduced VEGF protein expressionto levels approaching that of non-hypoxic HEK-293 cells. Anti-HIF-1alpha siRNA hHIF1#1 reduced VEGF protein expression to below that ofnon-hypoxic HEK-293 cells.

[0111] After the cell culture medium was removed from the control andexperimental cells, a cytotoxicity assay was performed as follows.Complete growth medium containing 10% AlamarBlue (Biosource, Camarillo,Calif.) was added to each well, and the cells were incubated at 37° C.with 5% CO₂ for 3 hours. Cell proliferation was measured by detectingthe color change of medium containing AlamarBlue resulting from cellmetabolic activity. Cytotoxicity assay results were read on an AD340plate reader (Beckman Coulter) and are given in FIG. 2. As can be seenfrom FIG. 2, none of the twenty anti-HIF-1 alpha siRNAs tested showedsignificant cytotoxicity in the HEK-293 cells.

[0112] After the cytotoxicity assay was performed, the growth medium ineach well was completely removed, and RNA extractions from the HEK-293cells were performed with the RNAqueous RNA isolation kit (Ambion,Austin, Tex.) according to the manufacturer's instructions. The levelsof human HIF-1 alpha and VEGF mRNA in the cells were measured byquantitative reverse transcription-polymerase chain reaction (RT-PCR),using the level of human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) mRNA as an internal standard.

[0113] The RT-PCR study showed that hypoxia increased the mRNA levels ofhuman VEGF relative to VEGF mRNA expression in non-hypoxic cells. TheVEGF mRNA levels in hypoxic cells were reduced by transfection withanti-HIF-1 alpha siRNAs. Transfection of hypoxic cells with non-specificsiRNA (EGFP siRNA) or mock transfection with no siRNA did not reduceVEGF mRNA levels. Thus, the introduction of anti-HIF-1 alpha siRNAs intothe HIK-293 cells induced the destruction of the VEGF mRNA, as comparedto cells transfected with non-specific siRNA or no siRNA. Thedestruction of VEGF mRNA induced by the anti-HIF-1 alpha siRNAscorrelated with the reduction in VEGF protein production shown in FIG.1.

EXAMPLE 2 In Vivo Inhibition of Angiogenesis with Anti-HIF-1 Alpha siRNAin a Mouse Model of Choroidal Neovascularization

[0114] Adult (8-15 week old) female C57B1/6 mice (n=7) were anesthetizedwith avertin (2,2,2-tribromoethanol) and their pupils were dilated with1% tropicamide. Laser photocoagulation was performed bilaterally using adiode laser photocoagulator (IRIS Medical, Mountain View, Calif.) and aslit lamp system with a cover slip as a contact lens. Laserphotocoagulation (140 mW; 75 micron spot size; 0.1 s duration) wasapplied to the 9, 12 and 3 o'clock positions in both eyes at 2 to 3 diskdiameters from the optic nerve. Since the rupture of Bruch's membrane isnecessary to create significant choroidal neovascularization (CNV),bubble formation at the time of photocoagulation was used as anindication of the rupture of Bruch's membrane. Laser burns that did notinduce a rupture in Bruch's membrane were excluded from the study.

[0115] Immediately after laser treatment, an siRNA targeted to mouseHIF-1 alpha mRNA was delivered to both eyes of each animal in the testgroup by intravitreal injection. Control animals received intravitrealinjection of carrier only.

[0116] The target sequence of the mouse anti-HIF-1 alpha mRNA wasAACTAACTGGACACAGTGTGT (SEQ ID NO: 297), and the siRNA used was:   5′-cuaacuggacacaguguguTT-3′ (SEQ ID NO: 298)3′ TTgauugaccugugucacaca5′ (SEQ ID NO: 299)

[0117] Twelve days after laser photocoagulation, the animals wereperfused with high molecular weight dextran-fluorescein (MolecularProbes, Eugene, Oreg.) to label the retinal/choroidal vasculature, andthe eyes were harvested. The area of each CNV was measured in choroidalflat mount preparations.

[0118] To prepare choroidal flat mounts, the anterior chamber wasremoved and the retina was extracted with the vitreous, leaving theeyecup. Relaxing incisions were made on the eye cup and the choroid wasflattened onto a slide. Using a Leica DMR microscope (Wetzlar, Germany)equipped with epifluorescence illumination, a masked investigatoridentified lesions in the dextran-fluorescein-perfused flat mountpreparations as circular fluorescent (fluorescein positive) areascorresponding to the area previously exposed to the laser light. Imagesof the lesions were captured using a black and white Hamamatsu CCDcamera (Hamamatsu Photonics, Bridgewater, N.J.) coupled to a AppleMacintosh G4 computer (Cupertino, Calif.) equipped with OpenLab 2.2software. Images for calibration were obtained from a slide with agrating of known size. The hyperfluorescent fluorescein-dextran labeledblood vessels within the area of the laser burn were selected as “regionof interest” (ROI) using Openlab software, and this software was used tocalculate the area (μm²) occupied by the white pixels in the ROIs. TheROIs were selected after collecting the images under identicalintegration settings by using the Openlab “magic wand” tool to identifypixels in the laser burn site at a range of 2000-4090 intensity units,as defined within the Openlab software. The intensity units which wereselected represented levels measured in normal fluorescein-perfusedvasculature. For reference, the intensity of background, non-fluorescentareas was <450 intensity units.

[0119] The ROIs were generally well-circumscribed by a region lackingfluorescence. After measuring the areas of CNV, images were colorized inOpenlab by applying an intensity ramp at 515 nanometers (the wavelengthat which the image data were captured), using the “apply wavelength”function in the Openlab software. This intensity ramp was applied to allof the pixels in the image, and made the whitest pixels the brightestgreen color. The images were then exported to Adobe Photoshop softwarefor presentation purposes. Situations in which there was no evidence ofa laser burn after bright field analysis of choroidal flatmounts wereexcluded.

[0120] Statistical analysis of the results was performed using aone-tailed distribution, two sample unequal variance Student's t-test.There was a statistically significant reduction in the CNV area(P=0.000354) between the anti-HIF-1 alpha siRNA treated animals and thecontrol lasered animals, indicating a substantial reduction inangiogenesis in the animals receiving the anti-HIF-1 alpha siRNA. Theresults are presented in FIG. 3.

1 299 1 2964 DNA Homo sapiens 1 ggccgtccct ggcggcggag atggcggcgacagcggcgga ggctgtgacc tctggctctg 60 gagagccccg ggaggaggct ggagccctcggccccgcctg gcatgaatcc cagttgcgca 120 gttatagctt cccgactagg cccattccgcgtctgagtca gagcgacccc cgggcagagg 180 agcttattga gaatgaggag cctgtggtgctgaccgacac aaatcttgtg tatcctgccc 240 tgaaatggga ccttgaatac ctgcaagagaatattggcaa tggagacttc tctgtgtaca 300 gtgccagcac ccacaagttc ttgtactatgatgagaagaa gatggccaat ttccagaact 360 ttaagccgag gtccaacagg gaagaaatgaaatttcatga gttcgttgag aaactgcagg 420 atatacagca gcgaggaggg gaagagaggttgtatctgca gcaaacgctc aatgacactg 480 tgggcgggaa gattgtcatg gacttcttaggttttaactg gaactggatt aataagcaac 540 agggaaagcg tggctggggg cagcttacctctaacctgct gctcattggc atggaaggaa 600 atgtgacacc tgctcactat gatgagcagcagaacttttt tgctcagata aaaggttaca 660 aacgatgcat cttattccct ccggatcagttcgagtgcct ctacccatac cctgttcatc 720 acccatgtga cagacagagc caggtggactttgacaatcc cgactacgag aggttcccta 780 atttccaaaa tgtggttggt tacgaaacagtggttggccc tggtgatgtt ctttacatcc 840 caatgtactg gtggcatcac atagagtcattactaaatgg ggggattacc atcactgtga 900 acttctggta taagggggct cccacccctaagagaattga atatcctctc aaagctcatc 960 agaaagtggc cataatgaga aacattgagaagatgcttgg agaggccttg gggaacccac 1020 aagaggtggg gcccttgttg aacacaatgatcaagggccg gtacaactag cctgccaggg 1080 gtcaaggcct cctgccaggt gactgctatcccgtccacac cgcttcattg atgaggacag 1140 gagactccaa gcgctagtat tgcacgctgcacttaatgga ctggactctt gccatggccc 1200 aggagtcagg tgtttggagc gaggcagggcagttggcact ccactcctat ttggagggac 1260 ttcataccct tgcctcttgt gccccagcaccttctctctc tgccccccgc ctaaagtcct 1320 gcattcagtg tgtggagtcc cagcttttggttgtcatcat gtctgtgtgt atgttagtct 1380 gtcaacttcg gaatgtgtgc gtgtgtgtgcatgcacacgc atgtatgtat ctgttccctg 1440 ttccttctgg gtcaggctgt cacttccggctctcggccct atctcctgca acctcagtgc 1500 ctcagcctga gagagagatg agatgctcttggactcccca ctgcatctgg gctgcagggc 1560 cagagctagt ctgaccatta ggtcagtctgcctcctgaca gtttttgcgt agtcaagctc 1620 taggcggtat gggaatggct accgggactctaatggggtg aaagagaggg gaggcttgcc 1680 tttgagagcc tatatagcct tcctgtgagagaggattaga tagggttcca actgggccta 1740 caagctcaag ccatacataa aaggaccttgggacataaga accaatgatt gtgcataagt 1800 tctaaattag agacacatat agtttctctctttcagcacc agctcttgcc cctatgctgg 1860 gtaccaaggg agttctccta gctgtggcttctctaggttc taggggtgca agcctctgtg 1920 tgtttgtttg tgtgtgtctg tgtgtgcgtatccacactag gggtgcaagc ctctgggtgt 1980 gtgtgtgtgt gtgcgtgcgt gtgtgtgtgtgtgtccgtgt gtgtgtgtgt gtgtccacac 2040 tggccagcct ccctacttac caaggttctccactgcttac cttttccagt gggacagtac 2100 agtgtgagcc cccgggaagt actgcctgacctatcctaag cttttacact tggattttag 2160 ccatcatatg ttggccaggt ttcactgcagcctgcccgag gctaactggc tagagcctcc 2220 aggccctatg atgctccctg cccaggccatatcctttatt cctgctgagc ttcctggctg 2280 aatagatgaa atggggtcaa gcccaggcagctcattcact atctgtgatc cacctcaggg 2340 cacgggcaaa cacataggct tgcgtcttaaagccagctcc tctgccagac cccgttgtaa 2400 tgtgccacaa caccctcaat agtcagggcaactggtggag catggaagtc gaatttcctt 2460 ttctgttagg agctactcct gggaacccctctcagggctg cagcttacag gtgggcagct 2520 gtgattgcac aacttgaagg gccatcattcacatctattc agtgggagtg gggtccctgg 2580 gattgggcag tgtggtggcc ctgtgtctcctcacctctgc tcctgtcttc atcaccttct 2640 ctctggaagg gaagaggagt tggaaggtctctggttttct tttctttttt tttttttgcc 2700 aaaggtttac ttccagcatc tgagctctggctctcacccc tgaagctcag ttatagtgca 2760 ctgatgaact gagaggatgc gtgtggatgtgtgtgcatgc ctgagtgcgt tttttgggga 2820 ggggtgttta tttttagtac cccattctggggttctctga tgcagtgtgg atgtgaagat 2880 atggtacctt ctcaagtgta gctctttcaaatatagtcaa tgctgggaaa aaaaaaaaaa 2940 aaaaaaaaaa aaaaaaaaaa aaaa 2964 23958 DNA Homo sapiens 2 gtgctgcctc gtctgagggg acaggaggat caccctcttcgtcgcttcgg ccagtgtgtc 60 gggctgggcc ctgacaagcc acctgaggag aggctcggagccgggcccgg accccggcga 120 ttgccgcccg cttctctcta gtctcacgag gggtttcccgcctcgcaccc ccacctctgg 180 acttgccttt ccttctcttc tccgcgtgtg gagggagccagcgcttaggc cggagcgagc 240 ctgggggccg cccgccgtga agacatcgcg gggaccgattcaccatggag ggcgccggcg 300 gcgcgaacga caagaaaaag ataagttctg aacgtcgaaaagaaaagtct cgagatgcag 360 ccagatctcg gcgaagtaaa gaatctgaag ttttttatgagcttgctcat cagttgccac 420 ttccacataa tgtgagttcg catcttgata aggcctctgtgatgaggctt accatcagct 480 atttgcgtgt gaggaaactt ctggatgctg gtgatttggatattgaagat gacatgaaag 540 cacagatgaa ttgcttttat ttgaaagcct tggatggttttgttatggtt ctcacagatg 600 atggtgacat gatttacatt tctgataatg tgaacaaatacatgggatta actcagtttg 660 aactaactgg acacagtgtg tttgatttta ctcatccatgtgaccatgag gaaatgagag 720 aaatgcttac acacagaaat ggccttgtga aaaagggtaaagaacaaaac acacagcgaa 780 gcttttttct cagaatgaag tgtaccctaa ctagccgaggaagaactatg aacataaagt 840 ctgcaacatg gaaggtattg cactgcacag gccacattcacgtatatgat accaacagta 900 accaacctca gtgtgggtat aagaaaccac ctatgacctgcttggtgctg atttgtgaac 960 ccattcctca cccatcaaat attgaaattc ctttagatagcaagactttc ctcagtcgac 1020 acagcctgga tatgaaattt tcttattgtg atgaaagaattaccgaattg atgggatatg 1080 agccagaaga acttttaggc cgctcaattt atgaatattatcatgctttg gactctgatc 1140 atctgaccaa aactcatcat gatatgttta ctaaaggacaagtcaccaca ggacagtaca 1200 ggatgcttgc caaaagaggt ggatatgtct gggttgaaactcaagcaact gtcatatata 1260 acaccaagaa ttctcaacca cagtgcattg tatgtgtgaattacgttgtg agtggtatta 1320 ttcagcacga cttgattttc tcccttcaac aaacagaatgtgtccttaaa ccggttgaat 1380 cttcagatat gaaaatgact cagctattca ccaaagttgaatcagaagat acaagtagcc 1440 tctttgacaa acttaagaag gaacctgatg ctttaactttgctggcccca gccgctggag 1500 acacaatcat atctttagat tttggcagca acgacacagaaactgatgac cagcaacttg 1560 aggaagtacc attatataat gatgtaatgc tcccctcacccaacgaaaaa ttacagaata 1620 taaatttggc aatgtctcca ttacccaccg ctgaaacgccaaagccactt cgaagtagtg 1680 ctgaccctgc actcaatcaa gaagttgcat taaaattagaaccaaatcca gagtcactgg 1740 aactttcttt taccatgccc cagattcagg atcagacacctagtccttcc gatggaagca 1800 ctagacaaag ttcacctgag cctaatagtc ccagtgaatattgtttttat gtggatagtg 1860 atatggtcaa tgaattcaag ttggaattgg tagaaaaactttttgctgaa gacacagaag 1920 caaagaaccc attttctact caggacacag atttagacttggagatgtta gctccctata 1980 tcccaatgga tgatgacttc cagttacgtt ccttcgatcagttgtcacca ttagaaagca 2040 gttccgcaag ccctgaaagc gcaagtcctc aaagcacagttacagtattc cagcagactc 2100 aaatacaaga acctactgct aatgccacca ctaccactgccaccactgat gaattaaaaa 2160 cagtgacaaa agaccgtatg gaagacatta aaatattgattgcatctcca tctcctaccc 2220 acatacataa agaaactact agtgccacat catcaccatatagagatact caaagtcgga 2280 cagcctcacc aaacagagca ggaaaaggag tcatagaacagacagaaaaa tctcatccaa 2340 gaagccctaa cgtgttatct gtcgctttga gtcaaagaactacagttcct gaggaagaac 2400 taaatccaaa gatactagct ttgcagaatg ctcagagaaagcgaaaaatg gaacatgatg 2460 gttcactttt tcaagcagta ggaattggaa cattattacagcagccagac gatcatgcag 2520 ctactacatc actttcttgg aaacgtgtaa aaggatgcaaatctagtgaa cagaatggaa 2580 tggagcaaaa gacaattatt ttaataccct ctgatttagcatgtagactg ctggggcaat 2640 caatggatga aagtggatta ccacagctga ccagttatgattgtgaagtt aatgctccta 2700 tacaaggcag cagaaaccta ctgcagggtg aagaattactcagagctttg gatcaagtta 2760 actgagcttt ttcttaattt cattcctttt tttggacactggtggctcac tacctaaagc 2820 agtctattta tattttctac atctaatttt agaagcctggctacaatact gcacaaactt 2880 ggttagttca atttttgatc ccctttctac ttaatttacattaatgctct tttttagtat 2940 gttctttaat gctggatcac agacagctca ttttctcagttttttggtat ttaaaccatt 3000 gcattgcagt agcatcattt taaaaaatgc acctttttatttatttattt ttggctaggg 3060 agtttatccc tttttcgaat tatttttaag aagatgccaatataattttt gtaagaaggc 3120 agtaaccttt catcatgatc ataggcagtt gaaaaatttttacacctttt ttttcacatt 3180 ttacataaat aataatgctt tgccagcagt acgtggtagccacaattgca caatatattt 3240 tcttaaaaaa taccagcagt tactcatgga atatattctgcgtttataaa actagttttt 3300 aagaagaaat tttttttggc ctatgaaatt gttaaacctggaacatgaca ttgttaatca 3360 tataataatg attcttaaat gctgtatggt ttattatttaaatgggtaaa gccatttaca 3420 taatatagaa agatatgcat atatctagaa ggtatgtggcatttatttgg ataaaattct 3480 caattcagag aaatcatctg atgtttctat agtcactttgccagctcaaa agaaaacaat 3540 accctatgta gttgtggaag tttatgctaa tattgtgtaactgatattaa acctaaatgt 3600 tctgcctacc ctgttggtat aaagatattt tgagcagactgtaaacaaga aaaaaaaaat 3660 catgcattct tagcaaaatt gcctagtatg ttaatttgctcaaaatacaa tgtttgattt 3720 tatgcacttt gtcgctatta acatcctttt tttcatgtagatttcaataa ttgagtaatt 3780 ttagaagcat tattttagga atatatagtt gtcacagtaaatatcttgtt ttttctatgt 3840 acattgtaca aatttttcat tccttttgct ctttgtggttggatctaaca ctaactgtat 3900 tgttttgtta catcaaataa acatcttctg tggaccaggaaaaaaaaaaa aaaaaaaa 3958 3 3812 DNA Homo sapiens 3 gtgctgcctc gtctgaggggacaggaggat caccctcttc gtcgcttcgg ccagtgtgtc 60 gggctgggcc ctgacaagccacctgaggag aggctcggag ccgggcccgg accccggcga 120 ttgccgcccg cttctctctagtctcacgag gggtttcccg cctcgcaccc ccacctctgg 180 acttgccttt ccttctcttctccgcgtgtg gagggagcca gcgcttaggc cggagcgagc 240 ctgggggccg cccgccgtgaagacatcgcg gggaccgatt caccatggag ggcgccggcg 300 gcgcgaacga caagaaaaagataagttctg aacgtcgaaa agaaaagtct cgagatgcag 360 ccagatctcg gcgaagtaaagaatctgaag ttttttatga gcttgctcat cagttgccac 420 ttccacataa tgtgagttcgcatcttgata aggcctctgt gatgaggctt accatcagct 480 atttgcgtgt gaggaaacttctggatgctg gtgatttgga tattgaagat gacatgaaag 540 cacagatgaa ttgcttttatttgaaagcct tggatggttt tgttatggtt ctcacagatg 600 atggtgacat gatttacatttctgataatg tgaacaaata catgggatta actcagtttg 660 aactaactgg acacagtgtgtttgatttta ctcatccatg tgaccatgag gaaatgagag 720 aaatgcttac acacagaaatggccttgtga aaaagggtaa agaacaaaac acacagcgaa 780 gcttttttct cagaatgaagtgtaccctaa ctagccgagg aagaactatg aacataaagt 840 ctgcaacatg gaaggtattgcactgcacag gccacattca cgtatatgat accaacagta 900 accaacctca gtgtgggtataagaaaccac ctatgacctg cttggtgctg atttgtgaac 960 ccattcctca cccatcaaatattgaaattc ctttagatag caagactttc ctcagtcgac 1020 acagcctgga tatgaaattttcttattgtg atgaaagaat taccgaattg atgggatatg 1080 agccagaaga acttttaggccgctcaattt atgaatatta tcatgctttg gactctgatc 1140 atctgaccaa aactcatcatgatatgttta ctaaaggaca agtcaccaca ggacagtaca 1200 ggatgcttgc caaaagaggtggatatgtct gggttgaaac tcaagcaact gtcatatata 1260 acaccaagaa ttctcaaccacagtgcattg tatgtgtgaa ttacgttgtg agtggtatta 1320 ttcagcacga cttgattttctcccttcaac aaacagaatg tgtccttaaa ccggttgaat 1380 cttcagatat gaaaatgactcagctattca ccaaagttga atcagaagat acaagtagcc 1440 tctttgacaa acttaagaaggaacctgatg ctttaacttt gctggcccca gccgctggag 1500 acacaatcat atctttagattttggcagca acgacacaga aactgatgac cagcaacttg 1560 aggaagtacc attatataatgatgtaatgc tcccctcacc caacgaaaaa ttacagaata 1620 taaatttggc aatgtctccattacccaccg ctgaaacgcc aaagccactt cgaagtagtg 1680 ctgaccctgc actcaatcaagaagttgcat taaaattaga accaaatcca gagtcactgg 1740 aactttcttt taccatgccccagattcagg atcagacacc tagtccttcc gatggaagca 1800 ctagacaaag ttcacctgagcctaatagtc ccagtgaata ttgtttttat gtggatagtg 1860 atatggtcaa tgaattcaagttggaattgg tagaaaaact ttttgctgaa gacacagaag 1920 caaagaaccc attttctactcaggacacag atttagactt ggagatgtta gctccctata 1980 tcccaatgga tgatgacttccagttacgtt ccttcgatca gttgtcacca ttagaaagca 2040 gttccgcaag ccctgaaagcgcaagtcctc aaagcacagt tacagtattc cagcagactc 2100 aaatacaaga acctactgctaatgccacca ctaccactgc caccactgat gaattaaaaa 2160 cagtgacaaa agaccgtatggaagacatta aaatattgat tgcatctcca tctcctaccc 2220 acatacataa agaaactactagtgccacat catcaccata tagagatact caaagtcgga 2280 cagcctcacc aaacagagcaggaaaaggag tcatagaaca gacagaaaaa tctcatccaa 2340 gaagccctaa cgtgttatctgtcgctttga gtcaaagaac tacagttcct gaggaagaac 2400 taaatccaaa gatactagctttgcagaatg ctcagagaaa gcgaaaaatg gaacatgatg 2460 gttcactttt tcaagcagtaggaattattt agcatgtaga ctgctggggc aatcaatgga 2520 tgaaagtgga ttaccacagctgaccagtta tgattgtgaa gttaatgctc ctatacaagg 2580 cagcagaaac ctactgcagggtgaagaatt actcagagct ttggatcaag ttaactgagc 2640 tttttcttaa tttcattcctttttttggac actggtggct cactacctaa agcagtctat 2700 ttatattttc tacatctaattttagaagcc tggctacaat actgcacaaa cttggttagt 2760 tcaatttttg atcccctttctacttaattt acattaatgc tcttttttag tatgttcttt 2820 aatgctggat cacagacagctcattttctc agttttttgg tatttaaacc attgcattgc 2880 agtagcatca ttttaaaaaatgcacctttt tatttattta tttttggcta gggagtttat 2940 ccctttttcg aattatttttaagaagatgc caatataatt tttgtaagaa ggcagtaacc 3000 tttcatcatg atcataggcagttgaaaaat ttttacacct tttttttcac attttacata 3060 aataataatg ctttgccagcagtacgtggt agccacaatt gcacaatata ttttcttaaa 3120 aaataccagc agttactcatggaatatatt ctgcgtttat aaaactagtt tttaagaaga 3180 aatttttttt ggcctatgaaattgttaaac ctggaacatg acattgttaa tcatataata 3240 atgattctta aatgctgtatggtttattat ttaaatgggt aaagccattt acataatata 3300 gaaagatatg catatatctagaaggtatgt ggcatttatt tggataaaat tctcaattca 3360 gagaaatcat ctgatgtttctatagtcact ttgccagctc aaaagaaaac aataccctat 3420 gtagttgtgg aagtttatgctaatattgtg taactgatat taaacctaaa tgttctgcct 3480 accctgttgg tataaagatattttgagcag actgtaaaca agaaaaaaaa aatcatgcat 3540 tcttagcaaa attgcctagtatgttaattt gctcaaaata caatgtttga ttttatgcac 3600 tttgtcgcta ttaacatcctttttttcatg tagatttcaa taattgagta attttagaag 3660 cattatttta ggaatatatagttgtcacag taaatatctt gttttttcta tgtacattgt 3720 acaaattttt cattccttttgctctttgtg gttggatcta acactaactg tattgttttg 3780 ttacatcaaa taaacatcttctgtggacca gg 3812 4 3718 DNA Rattus norvegicus 4 gacaccgcgg gcaccgattcgccatggagg gcgccggcgg cgagaacgag aagaaaaata 60 ggatgagttc cgaacgtcgaaaagaaaagt ctagggatgc agcacgatct cggcgaagca 120 aagagtctga agttttttatgagcttgctc atcagttgcc acttccccac aacgtgagct 180 cccatcttga taaagcttctgttatgaggc tcaccatcag ttacttacgt gtgaggaaac 240 ttctaggtgc tggtgatcttgacattgaag atgaaatgaa agcacagatg aactgctttt 300 atctgaaagc cctggatggctttgttatgg tgctaacaga tgatggtgac atgatttaca 360 tttctgataa cgtgaacaaatacatggggt tgactcagtt tgaactaact ggacacagtg 420 tgtttgattt tacccatccatgtgaccatg aggaaatgag agaaatgctt acacacagaa 480 atggcccagt gagaaaggggaaagaacaaa acacgcagcg aagctttttt ctcagaatga 540 aatgtaccct aacaagccgggggaggacga tgaacatcaa gtcagcaacg tggaaggtgc 600 tgcactgcac aggccacattcatgtgtatg ataccagcag taaccagccg cagtgtggct 660 acaagaaacc gcctatgacgtgcttggtgc tgatttgtga acccattcct catccatcaa 720 acattgaaat tcctttagacagcaagacat ttctcagtcg acacagcctc gatatgaaat 780 tttcttactg tgatgaaaggattactgagt tgatgggtta tgagccagaa gaacttttgg 840 gccgttcaat ttatgaatattatcatgctt tggactctga tcatctgacc aaaactcatc 900 atgacatgtt tactaaaggacaagtcacca caggacagta caggatgctt gcaaaaagag 960 gtggatatgt ctgggttgagactcaagcaa ctgttatata taatacgaag aactctcagc 1020 cacagtgcat tgtgtgtgtgaattatgttg taagtggtat tattcagcac gacttgattt 1080 tctcccttca acaaacagaatctgtcctca aaccagttga atcttcagat atgaaaatga 1140 cccagctgtt cactaaagtggaatctgagg acacgagctg cctcttcgac aagcttaaga 1200 aagagcccga tgccctgactctgctagctc cagcggctgg ggacacgatc atatcactgg 1260 acttcggcag cgatgacacggaaactgaag accaacaact tgaagatgtc ccgttgtaca 1320 atgatgtaat gttcccctcttctaatgaga aattaaatat aaatctggca atgtctccat 1380 tacctgcctc tgaaactccaaagccacttc gaagtagtgc tgatcctgca ctgaatcaag 1440 aggttgcatt gaagttagagtcaagcccag agtcactggg actttctttt accatgcccc 1500 agattcaaga tcagccagcaagtccttctg atggaagcac tagacaaagc tcacctgagc 1560 ctaacagtcc cagtgagtactgctttgatg tggacagcga tatggtcaat gtattcaagt 1620 tggaactggt ggaaaaactgtttgctgaag acacagaagc gaagaatcca ttttcagctc 1680 aggacactga tttagacttggaaatgctgg ctccctatat cccaatggat gatgatttcc 1740 agttacgttc ctttgatcagttgtcaccat tagagagcaa ttctccaagc cctccgagtg 1800 tgagcacagt tacaggattccagcagaccc agttacagaa acctaccatc actgtcactg 1860 ccaccgcaac tgccaccactgatgaatcaa aagcagtgac gaaggacaat atagaagaca 1920 ttaaaatact gattgcatctccaccttcta cccaagtacc tcaagaaatg accactgcta 1980 aggcatcagc atacagtggtactcacagtc ggacagcctc accagacaga gcaggaaaga 2040 gagtcataga aaaaacagacaaagctcatc caaggagcct taacctatct gtcactttga 2100 atcaaagaaa tactgttcctgaagaagaat taaacccaaa gacaatagct ttgcagaatg 2160 ctcagaggaa gcgaaaaatggaacatgatg gctccctttt tcaagcagca ggaattggaa 2220 cgttactgca gcaaccaggtgaccgtgccc ctactatgtc gctttcttgg aaacgagtga 2280 aaggatacat atctagtgaacaggatggaa tggagcagaa gacaattttt ttaataccct 2340 ctgatttagc atgtagactgctggggcagt caatggatga gagtggatta ccacagctga 2400 ccagttacga ttgtgaagttaatgctccca tacaaggcag cagaaaccta ctgcagggtg 2460 aagaattact cagagctttggatcaagtta actgagcttt tcctaatctc attcctttga 2520 ttgttaattt ttgtgttcagttgttgttgt tgtctgtggg gtttcgtttc tgttggttgt 2580 tttggacact ggtggctcagcagtctattt atattttcta tatctcattt agaggcctgg 2640 ctacagtact gcaccaactcagatagttta gtttgggccc cttcctcctt cattttcact 2700 gatgctcttt ttaccatgtccttcgaatgc cagatcacag cacattcaca gctccccagc 2760 atttcaccaa tgcattgctgtagtgtcgtt taaaatgcac ctttttattt atttattttt 2820 ggtgagggag tttgtcccttattgaattat ttttaatgaa atgccaatat aattttttaa 2880 gaaggcagta aatcttcatcatgatgatag gcagttgaaa attttttact catttttttc 2940 atgttttaca tgaaaataatgctttgccag cagtacatgg tagccacaat tgcacaatat 3000 attttcttaa aaataccagcagttactcat gcatatattc tgcatttata aaactagttt 3060 ttaagaagaa actttttttggcctatggaa ttgttaagcc tggatcatga tgctgttgat 3120 cttataatga ttcttaaactgtatggtttc tttatatggg taaagccatt tacatgatat 3180 agagagatat gcttatatctggaaggtata tggcatttat ttggataaaa ttctcaattg 3240 agaagttatc tggtgtttctttactttacc ggctcaaaag aaaacagtcc ctatgtagtt 3300 gtggaagctt atgctaatattgtgtaattg atattaaaca ttaaatgttc tgcctatcct 3360 gttggtataa agacattttgagcatactgt aaacaaaaaa atcatgcatt gttagtaaaa 3420 ttgcctagta tgttaatttgttgaaaatac gatgtttggt tttatgcact ttgtcgctat 3480 taacatcctt tttttcatatagatttcaat aattgagtaa ttttagaagc attattttag 3540 aaatatagag ttgtcatagtaaacatcttg tttttttttc tttttttcta tgtacattgt 3600 ataaattttt cattcccttgctctttgtag ttgggtctaa cactaactgt actgttttgt 3660 tatatcaaat aaacatcttctgtggaccag gaaaaaaaaa aaaaaaaaaa aaaaaaaa 3718 5 3973 DNA Mus musculus 5cgcgaggact gtcctcgccg ccgtcgcggg cagtgtctag ccaggccttg acaagctagc 60cggaggagcg cctaggaacc cgagccggag ctcagcgagc gcagcctgca cgcccgcctc 120gcgtcccggg ggggtcccgc ctcccacccc gcctctggac ttgtctcttt ccccgcgcgc 180gcggacagag ccggcgttta ggcccgagcg agcccggggg ccgccggccg ggaagacaac 240gcgggcaccg attcgccatg gagggcgccg gcggcgagaa cgagaagaaa aagatgagtt 300ctgaacgtcg aaaagaaaag tctagagatg cagcaagatc tcggcgaagc aaagagtctg 360aagtttttta tgagcttgct catcagttgc cacttcccca caatgtgagc tcacatcttg 420ataaagcttc tgttatgagg ctcaccatca gttatttacg tgtgagaaaa cttctggatg 480ccggtggtct agacagtgaa gatgagatga aggcacagat ggactgtttt tatctgaaag 540ccctagatgg ctttgtgatg gtgctaacag atgacggcga catggtttac atttctgata 600acgtgaacaa atacatgggg ttaactcagt ttgaactaac tggacacagt gtgtttgatt 660ttactcatcc atgtgaccat gaggaaatga gagaaatgct tacacacaga aatggcccag 720tgagaaaagg gaaagaacta aacacacagc ggagcttttt tctcagaatg aagtgcaccc 780taacaagccg ggggaggacg atgaacatca agtcagcaac gtggaaggtg cttcactgca 840cgggccatat tcatgtctat gataccaaca gtaaccaacc tcagtgtggg tacaagaaac 900cacccatgac gtgcttggtg ctgatttgtg aacccattcc tcatccgtca aatattgaaa 960ttcctttaga tagcaagaca tttctcagtc gacacagcct cgatatgaaa ttttcttact 1020gtgatgaaag aattactgag ttgatgggtt atgagccgga agaacttttg ggccgctcaa 1080tttatgaata ttatcatgct ttggattctg atcatctgac caaaactcac catgatatgt 1140ttactaaagg acaagtcacc acaggacagt acaggatgct tgccaaaaga ggtggatatg 1200tctgggttga aactcaagca actgtcatat ataatacgaa gaactcccag ccacagtgca 1260ttgtgtgtgt gaattatgtt gtaagtggta ttattcagca cgacttgatt ttctcccttc 1320aacaaacaga atctgtgctc aaaccagttg aatcttcaga tatgaagatg actcagctgt 1380tcaccaaagt tgaatcagag gatacaagct gcctttttga taagcttaag aaggagcctg 1440atgctctcac tctgctggct ccagctgccg gcgacaccat catctctctg gattttggca 1500gcgatgacac agaaactgaa gatcaacaac ttgaagatgt tccattatat aatgatgtaa 1560tgtttccctc ttctaatgaa aaattaaata taaacctggc aatgtctcct ttaccttcat 1620cggaaactcc aaagccactt cgaagtagtg ctgatcctgc actgaatcaa gaggttgcat 1680taaaattaga atcaagtcca gagtcactgg gactttcttt taccatgccc cagattcaag 1740atcagccagc aagtccttct gatggaagca ctagacaaag ttcacctgag agacttcttc 1800aggaaaacgt aaacactcct aacttttccc agcctaacag tcccagtgaa tattgctttg 1860atgtggatag cgatatggtc aatgtattca agttggaact ggtggaaaaa ctgtttgctg 1920aagacacaga ggcaaagaat ccattttcaa ctcaggacac tgatttagat ttggagatgc 1980tggctcccta tatcccaatg gatgatgatt tccagttacg ttcctttgat cagttgtcac 2040cattagagag caattctcca agccctccaa gtatgagcac agttactggg ttccagcaga 2100cccagttaca gaaacctacc atcactgcca ctgccaccac aactgccacc actgatgaat 2160caaaaacaga gacgaaggac aataaagaag atattaaaat actgattgca tctccatctt 2220ctacccaagt acctcaagaa acgaccactg ctaaggcatc agcatacagt ggcactcaca 2280gtcggacagc ctcaccagac agagcaggaa agagagtcat agaacagaca gacaaagctc 2340atccaaggag ccttaagctg tctgccactt tgaatcaaag aaatactgtt cctgaggaag 2400aattaaaccc aaagacaata gcttcgcaga atgctcagag gaagcgaaaa atggaacatg 2460atggctccct ttttcaagca gcaggaattg gaacattatt gcagcaacca ggtgactgtg 2520cacctactat gtcactttcc tggaaacgag tgaaaggatt catatctagt gaacagaatg 2580gaacggagca aaagactatt attttaatac cctccgattt agcatgcaga ctgctggggc 2640agtcaatgga tgagagtgga ttaccacagc tgaccagtta cgattgtgaa gttaatgctc 2700ccatacaagg cagcagaaac ctactgcagg gtgaagaatt actcagagct ttggatcaag 2760ttaactgagc gtttcctaat ctcattcctt ttgattgtta atgtttttgt tcagttgttg 2820ttgtttgttg ggtttttgtt tctgttggtt atttttggac actggtggct cagcagtcta 2880tttatatttt ctatatctaa ttttagaagc ctggctacaa tactgcacaa actcagatag 2940tttagttttc atcccctttc tacttaattt tcattaatgc tctttttaat atgttctttt 3000aatgccagat cacagcacat tcacagctcc tcagcatttc accattgcat tgctgtagtg 3060tcatttaaaa tgcacctttt tatttattta tttttggtga gggagtttgt cccttattga 3120attattttta atgaaatgcc aatataattt tttaagaaag cagtaaattc tcatcatgat 3180cataggcagt tgaaaacttt ttactcattt ttttcatgtt ttacatgaaa ataatgcttt 3240gtcagcagta catggtagcc acaattgcac aatatatttt ctttaaaaaa ccagcagtta 3300ctcatgcaat atattctgca tttataaaac tagtttttaa gaaatttttt ttggcctatg 3360gaattgttaa gcctggatca tgaagcgttg atcttataat gattcttaaa ctgtatggtt 3420tctttatatg ggtaaagcca tttacatgat ataaagaaat atgcttatat ctggaaggta 3480tgtggcattt atttggataa aattctcaat tcagagaagt tatctggtgt ttcttgactt 3540taccaactca aaacagtccc tctgtagttg tggaagctta tgctaatatt gtgtaattga 3600ttatgaaaca taaatgttct gcccaccctg ttggtataaa gacattttga gcatactgta 3660aacaaacaaa caaaaaatca tgctttgtta gtaaaattgc ctagtatgtt gatttgttga 3720aaatatgatg tttggtttta tgcactttgt cgctattaac atcctttttt catatagatt 3780tcaataagtg agtaatttta gaagcattat tttaggaata tagagttgtc atagtaaaca 3840tcttgttttt tctatgtaca ctgtataaat ttttcgttcc cttgctcttt gtggttgggt 3900ctaacactaa ctgtactgtt ttgttatatc aaataaacat cttctgtgga ccaggaaaaa 3960aaaaaaaaaa aaa 3973 6 21 DNA Artificial Sequence target sequence 6aactggacac agtgtgtttg a 21 7 23 RNA Artificial Sequence siRNA sensestrand 7 aacuaacugg acacagugug uuu 23 8 23 RNA Artificial Sequence siRNAantisense strand 8 acacacugug uccaguuagu uuu 23 9 23 DNA ArtificialSequence siRNA sense strand 9 aacuaacugg acacagugug utt 23 10 23 DNAArtificial Sequence siRNA antisense strand 10 acacacugug uccaguuagu utt23 11 21 DNA Artificial Sequence target sequence 11 aactaactggacacagtgtg t 21 12 17 DNA Artificial Sequence target sequence 12cgacaagaaa aagataa 17 13 17 DNA Artificial Sequence target sequence 13aaagataagt tctgaac 17 14 17 DNA Artificial Sequence target sequence 14agataagttc tgaacgt 17 15 17 DNA Artificial Sequence target sequence 15gttctgaacg tcgaaaa 17 16 17 DNA Artificial Sequence target sequence 16aagaaaagtc tcgagat 17 17 17 DNA Artificial Sequence target sequence 17gaaaagtctc gagatgc 17 18 17 DNA Artificial Sequence target sequence 18agtctcgaga tgcagcc 17 19 17 DNA Artificial Sequence target sequence 19gtaaagaatc tgaagtt 17 20 17 DNA Artificial Sequence target sequence 20gaatctgaag tttttta 17 21 17 DNA Artificial Sequence target sequence 21gttttttatg agcttgc 17 22 17 DNA Artificial Sequence target sequence 22ggcctctgtg atgaggc 17 23 17 DNA Artificial Sequence target sequence 23cttctggatg ctggtga 17 24 17 DNA Artificial Sequence target sequence 24agcacagatg aattgct 17 25 21 DNA Artificial Sequence target sequence 25aaatgcttac acacagaaat g 21 26 17 DNA Artificial Sequence target sequence26 gaaaaagata agttctg 17 27 17 DNA Artificial Sequence target sequence27 aagataagtt ctgaacg 17 28 17 DNA Artificial Sequence target sequence28 gataagttct gaacgtc 17 29 17 DNA Artificial Sequence target sequence29 cgtcgaaaag aaaagtc 17 30 17 DNA Artificial Sequence target sequence30 agaaaagtct cgagatg 17 31 17 DNA Artificial Sequence target sequence31 aagtctcgag atgcagc 17 32 17 DNA Artificial Sequence target sequence32 gtctcgagat gcagcca 17 33 17 DNA Artificial Sequence target sequence33 agaatctgaa gtttttt 17 34 17 DNA Artificial Sequence target sequence34 tctgaagttt tttatga 17 35 17 DNA Artificial Sequence target sequence35 tgtgagttcg catcttg 17 36 17 DNA Artificial Sequence target sequence36 acttctggat gctggtg 17 37 17 DNA Artificial Sequence target sequence37 gatgacatga aagcaca 17 38 17 DNA Artificial Sequence target sequence38 gcacagatga attgctt 17 39 21 DNA Artificial Sequence target sequence39 aagtttttta tgagcttgct c 21 40 21 DNA Artificial Sequence targetsequence 40 aagtttttta tgagcttgct c 21 41 21 DNA Artificial Sequencetarget sequence 41 aaggcctctg tgatgaggct t 21 42 21 DNA ArtificialSequence target sequence 42 aaacttctgg atgctggtga t 21 43 21 DNAArtificial Sequence target sequence 43 aacttctgga tgctggtgat t 21 44 21DNA Artificial Sequence target sequence 44 aagatgacat gaaagcacag a 21 4521 DNA Artificial Sequence target sequence 45 aaagcacaga tgaattgctt t 2146 21 DNA Artificial Sequence target sequence 46 aagcacagat gaattgcttt t21 47 21 DNA Artificial Sequence target sequence 47 aattgcttttatttgaaagc c 21 48 21 DNA Artificial Sequence target sequence 48aaagccttgg atggttttgt t 21 49 21 DNA Artificial Sequence target sequence49 aagccttgga tggttttgtt a 21 50 21 DNA Artificial Sequence targetsequence 50 aatgtgaaca aatacatggg a 21 51 21 DNA Artificial Sequencetarget sequence 51 aacaaataca tgggattaac t 21 52 21 DNA ArtificialSequence target sequence 52 aaatacatgg gattaactca g 21 53 21 DNAArtificial Sequence target sequence 53 aaatacatgg gattaactca g 21 54 21DNA Artificial Sequence target sequence 54 aactcagttt gaactaactg g 21 5521 DNA Artificial Sequence target sequence 55 aactaactgg acacagtgtg t 2156 21 DNA Artificial Sequence target sequence 56 aactggacac agtgtgtttg a21 57 21 DNA Artificial Sequence target sequence 57 aaatgagagaaatgcttaca c 21 58 21 DNA Artificial Sequence target sequence 58aatgagagaa atgcttacac a 21 59 21 DNA Artificial Sequence target sequence59 aaatgcttac acacagaaat g 21 60 21 DNA Artificial Sequence targetsequence 60 aatgcttaca cacagaaatg g 21 61 21 DNA Artificial Sequencetarget sequence 61 aaatggcctt gtgaaaaagg g 21 62 21 DNA ArtificialSequence target sequence 62 aatggccttg tgaaaaaggg t 21 63 21 DNAArtificial Sequence target sequence 63 aaaaagggta aagaacaaaa c 21 64 21DNA Artificial Sequence target sequence 64 aaaagggtaa agaacaaaac a 21 6521 DNA Artificial Sequence target sequence 65 aaagggtaaa gaacaaaaca c 2166 21 DNA Artificial Sequence target sequence 66 aagggtaaag aacaaaacac a21 67 21 DNA Artificial Sequence target sequence 67 aaagaacaaaacacacagcg a 21 68 21 DNA Artificial Sequence target sequence 68aagaacaaaa cacacagcga a 21 69 21 DNA Artificial Sequence target sequence69 aacaaaacac acagcgaagc t 21 70 21 DNA Artificial Sequence targetsequence 70 aacaaaacac acagcgaagc t 21 71 21 DNA Artificial Sequencetarget sequence 71 aaacacacag cgaagctttt t 21 72 21 DNA ArtificialSequence target sequence 72 aacacacagc gaagcttttt t 21 73 21 DNAArtificial Sequence target sequence 73 aagctttttt ctcagaatga a 21 74 21DNA Artificial Sequence target sequence 74 aatgaagtgt accctaacta g 21 7521 DNA Artificial Sequence target sequence 75 aagtgtaccc taactagccg a 2176 21 DNA Artificial Sequence target sequence 76 aactagccga ggaagaacta t21 77 21 DNA Artificial Sequence target sequence 77 aagaactatgaacataaagt c 21 78 21 DNA Artificial Sequence target sequence 78aactatgaac ataaagtctg c 21 79 21 DNA Artificial Sequence target sequence79 aacataaagt ctgcaacatg g 21 80 21 DNA Artificial Sequence targetsequence 80 aaagtctgca acatggaagg t 21 81 21 DNA Artificial Sequencetarget sequence 81 aagtctgcaa catggaaggt a 21 82 21 DNA ArtificialSequence target sequence 82 aacatggaag gtattgcact g 21 83 21 DNAArtificial Sequence target sequence 83 aaggtattgc actgcacagg c 21 84 21DNA Artificial Sequence target sequence 84 aacagtaacc aacctcagtg t 21 8521 DNA Artificial Sequence target sequence 85 aaccaacctc agtgtgggta t 2186 21 DNA Artificial Sequence target sequence 86 aacctcagtg tgggtataag a21 87 21 DNA Artificial Sequence target sequence 87 aagaaaccacctatgacctg c 21 88 21 DNA Artificial Sequence target sequence 88aagaaaccac ctatgacctg c 21 89 21 DNA Artificial Sequence target sequence89 aaccacctat gacctgcttg g 21 90 21 DNA Artificial Sequence targetsequence 90 aacccattcc tcacccatca a 21 91 21 DNA Artificial Sequencetarget sequence 91 aaatattgaa attcctttag a 21 92 21 DNA ArtificialSequence target sequence 92 aatattgaaa ttcctttaga t 21 93 21 DNAArtificial Sequence target sequence 93 aaattccttt agatagcaag a 21 94 21DNA Artificial Sequence target sequence 94 aattccttta gatagcaaga c 21 9521 DNA Artificial Sequence target sequence 95 aagactttcc tcagtcgaca c 2196 21 DNA Artificial Sequence target sequence 96 aaattttctt attgtgatga a21 97 21 DNA Artificial Sequence target sequence 97 aattttcttattgtgatgaa a 21 98 21 DNA Artificial Sequence target sequence 98aaagaattac cgaattgatg g 21 99 21 DNA Artificial Sequence target sequence99 aattaccgaa ttgatgggat a 21 100 21 DNA Artificial Sequence targetsequence 100 aattaccgaa ttgatgggat a 21 101 21 DNA Artificial Sequencetarget sequence 101 aagaactttt aggccgctca a 21 102 21 DNA ArtificialSequence target sequence 102 aacttttagg ccgctcaatt t 21 103 21 DNAArtificial Sequence target sequence 103 aatttatgaa tattatcatg c 21 10421 DNA Artificial Sequence target sequence 104 aatattatca tgctttggac t21 105 21 DNA Artificial Sequence target sequence 105 aaaactcatcatgatatgtt t 21 106 21 DNA Artificial Sequence target sequence 106aaactcatca tgatatgttt a 21 107 21 DNA Artificial Sequence targetsequence 107 aactcatcat gatatgttta c 21 108 21 DNA Artificial Sequencetarget sequence 108 aaaggacaag tcaccacagg a 21 109 21 DNA ArtificialSequence target sequence 109 aaggacaagt caccacagga c 21 110 21 DNAArtificial Sequence target sequence 110 aagtcaccac aggacagtac a 21 11121 DNA Artificial Sequence target sequence 111 aaaagaggtg gatatgtctg g21 112 21 DNA Artificial Sequence target sequence 112 aaagaggtggatatgtctgg g 21 113 21 DNA Artificial Sequence target sequence 113aagaggtgga tatgtctggg t 21 114 21 DNA Artificial Sequence targetsequence 114 aaactcaagc aactgtcata t 21 115 21 DNA Artificial Sequencetarget sequence 115 aactcaagca actgtcatat a 21 116 21 DNA ArtificialSequence target sequence 116 aagcaactgt catatataac a 21 117 21 DNAArtificial Sequence target sequence 117 aactgtcata tataacacca a 21 11821 DNA Artificial Sequence target sequence 118 aacaccaaga attctcaacc a21 119 21 DNA Artificial Sequence target sequence 119 aagaattctcaaccacagtg c 21 120 21 DNA Artificial Sequence target sequence 120aattctcaac cacagtgcat t 21 121 21 DNA Artificial Sequence targetsequence 121 aaccacagtg cattgtatgt g 21 122 21 DNA Artificial Sequencetarget sequence 122 aattacgttg tgagtggtat t 21 123 21 DNA ArtificialSequence target sequence 123 aacaaacaga atgtgtcctt a 21 124 21 DNAArtificial Sequence target sequence 124 aaacagaatg tgtccttaaa c 21 12521 DNA Artificial Sequence target sequence 125 aacagaatgt gtccttaaac c21 126 20 DNA Artificial Sequence target sequence 126 atgtgtccttaaaccggttg 20 127 21 DNA Artificial Sequence target sequence 127aaaccggttg aatcttcaga t 21 128 21 DNA Artificial Sequence targetsequence 128 aaccggttga atcttcagat a 21 129 21 DNA Artificial Sequencetarget sequence 129 aatcttcaga tatgaaaatg a 21 130 21 DNA ArtificialSequence target sequence 130 aaaatgactc agctattcac c 21 131 21 DNAArtificial Sequence target sequence 131 aaatgactca gctattcacc a 21 13221 DNA Artificial Sequence target sequence 132 aatgactcag ctattcacca a21 133 21 DNA Artificial Sequence target sequence 133 aaagttgaatcagaagatac a 21 134 21 DNA Artificial Sequence target sequence 134aagttgaatc agaagataca a 21 135 21 DNA Artificial Sequence targetsequence 135 aatcagaaga tacaagtagc c 21 136 21 DNA Artificial Sequencetarget sequence 136 aagatacaag tagcctcttt g 21 137 21 DNA ArtificialSequence target sequence 137 aagtagcctc tttgacaaac t 21 138 21 DNAArtificial Sequence target sequence 138 aaacttaaga aggaacctga t 21 13921 DNA Artificial Sequence target sequence 139 aacttaagaa ggaacctgat g21 140 21 DNA Artificial Sequence target sequence 140 aagaaggaacctgatgcttt a 21 141 21 DNA Artificial Sequence target sequence 141aaggaacctg atgctttaac t 21 142 21 DNA Artificial Sequence targetsequence 142 aacctgatgc tttaactttg c 21 143 21 DNA Artificial Sequencetarget sequence 143 aactttgctg gccccagccg c 21 144 21 DNA ArtificialSequence target sequence 144 aatcatatct ttagattttg g 21 145 21 DNAArtificial Sequence target sequence 145 aacgacacag aaactgatga c 21 14621 DNA Artificial Sequence target sequence 146 aaactgatga ccagcaactt g21 147 21 DNA Artificial Sequence target sequence 147 aactgatgaccagcaacttg a 21 148 21 DNA Artificial Sequence target sequence 148aacttgagga agtaccatta t 21 149 21 DNA Artificial Sequence targetsequence 149 aagtaccatt atataatgat g 21 150 21 DNA Artificial Sequencetarget sequence 150 aatgatgtaa tgctcccctc a 21 151 21 DNA ArtificialSequence target sequence 151 aatgctcccc tcacccaacg a 21 152 21 DNAArtificial Sequence target sequence 152 aacgaaaaat tacagaatat a 21 15321 DNA Artificial Sequence target sequence 153 aaaaattaca gaatataaat t21 154 21 DNA Artificial Sequence target sequence 154 aaaattacagaatataaatt t 21 155 21 DNA Artificial Sequence target sequence 155aaattacaga atataaattt g 21 156 21 DNA Artificial Sequence targetsequence 156 aattacagaa tataaatttg g 21 157 21 DNA Artificial Sequencetarget sequence 157 aatataaatt tggcaatgtc t 21 158 21 DNA ArtificialSequence target sequence 158 aaatttggca atgtctccat t 21 159 21 DNAArtificial Sequence target sequence 159 aatttggcaa tgtctccatt a 21 16021 DNA Artificial Sequence target sequence 160 aatgtctcca ttacccaccg c21 161 21 DNA Artificial Sequence target sequence 161 aaacgccaaagccacttcga a 21 162 21 DNA Artificial Sequence target sequence 162aacgccaaag ccacttcgaa g 21 163 21 DNA Artificial Sequence targetsequence 163 aaagccactt cgaagtagtg c 21 164 21 DNA Artificial Sequencetarget sequence 164 aagccacttc gaagtagtgc t 21 165 21 DNA ArtificialSequence target sequence 165 aagtagtgct gaccctgcac t 21 166 21 DNAArtificial Sequence target sequence 166 aatcaagaag ttgcattaaa a 21 16721 DNA Artificial Sequence target sequence 167 aagaagttgc attaaaatta g21 168 21 DNA Artificial Sequence target sequence 168 aagttgcattaaaattagaa c 21 169 21 DNA Artificial Sequence target sequence 169aaaattagaa ccaaatccag a 21 170 21 DNA Artificial Sequence targetsequence 170 aaattagaac caaatccaga g 21 171 21 DNA Artificial Sequencetarget sequence 171 aattagaacc aaatccagag t 21 172 21 DNA ArtificialSequence target sequence 172 aaccaaatcc agagtcactg g 21 173 21 DNAArtificial Sequence target sequence 173 aaatccagag tcactggaac t 21 17421 DNA Artificial Sequence target sequence 174 aatccagagt cactggaact t21 175 21 DNA Artificial Sequence target sequence 175 aactttcttttaccatgccc c 21 176 21 DNA Artificial Sequence target sequence 176aagcactaga caaagttcac c 21 177 21 DNA Artificial Sequence targetsequence 177 aaagttcacc tgagcctaat a 21 178 21 DNA Artificial Sequencetarget sequence 178 aagttcacct gagcctaata g 21 179 21 DNA ArtificialSequence target sequence 179 aatagtccca gtgaatattg t 21 180 21 DNAArtificial Sequence target sequence 180 aatattgttt ttatgtggat a 21 18121 DNA Artificial Sequence target sequence 181 aatgaattca agttggaatt g21 182 21 DNA Artificial Sequence target sequence 182 aattcaagttggaattggta g 21 183 21 DNA Artificial Sequence target sequence 183aagttggaat tggtagaaaa a 21 184 21 DNA Artificial Sequence targetsequence 184 aattggtaga aaaacttttt g 21 185 21 DNA Artificial Sequencetarget sequence 185 aaaacttttt gctgaagaca c 21 186 21 DNA ArtificialSequence target sequence 186 aaactttttg ctgaagacac a 21 187 21 DNAArtificial Sequence target sequence 187 aactttttgc tgaagacaca g 21 18821 DNA Artificial Sequence target sequence 188 aagacacaga agcaaagaac c21 189 21 DNA Artificial Sequence target sequence 189 aagcaaagaacccattttct a 21 190 21 DNA Artificial Sequence target sequence 190aaagaaccca ttttctactc a 21 191 21 DNA Artificial Sequence targetsequence 191 aagaacccat tttctactca g 21 192 21 DNA Artificial Sequencetarget sequence 192 aacccatttt ctactcagga c 21 193 21 DNA ArtificialSequence target sequence 193 aatggatgat gacttccagt t 21 194 21 DNAArtificial Sequence target sequence 194 aaagcagttc cgcaagccct g 21 19521 DNA Artificial Sequence target sequence 195 aagcagttcc gcaagccctg a21 196 21 DNA Artificial Sequence target sequence 196 aagccctgaaagcgcaagtc c 21 197 21 DNA Artificial Sequence target sequence 197aaagcgcaag tcctcaaagc a 21 198 21 DNA Artificial Sequence targetsequence 198 aagcgcaagt cctcaaagca c 21 199 21 DNA Artificial Sequencetarget sequence 199 aagtcctcaa agcacagtta c 21 200 21 DNA ArtificialSequence target sequence 200 aaagcacagt tacagtattc c 21 201 21 DNAArtificial Sequence target sequence 201 aagcacagtt acagtattcc a 21 20221 DNA Artificial Sequence target sequence 202 aaatacaaga acctactgct a21 203 21 DNA Artificial Sequence target sequence 203 aatacaagaacctactgcta a 21 204 21 DNA Artificial Sequence target sequence 204aagaacctac tgctaatgcc a 21 205 21 DNA Artificial Sequence targetsequence 205 aacctactgc taatgccacc a 21 206 21 DNA Artificial Sequencetarget sequence 206 aatgccacca ctaccactgc c 21 207 21 DNA ArtificialSequence target sequence 207 aattaaaaac agtgacaaaa g 21 208 21 DNAArtificial Sequence target sequence 208 aaaaacagtg acaaaagacc g 21 20921 DNA Artificial Sequence target sequence 209 aaaacagtga caaaagaccg t21 210 21 DNA Artificial Sequence target sequence 210 aaacagtgacaaaagaccgt a 21 211 21 DNA Artificial Sequence target sequence 211aacagtgaca aaagaccgta t 21 212 21 DNA Artificial Sequence targetsequence 212 aaaagaccgt atggaagaca t 21 213 21 DNA Artificial Sequencetarget sequence 213 aaagaccgta tggaagacat t 21 214 21 DNA ArtificialSequence target sequence 214 aagaccgtat ggaagacatt a 21 215 21 DNAArtificial Sequence target sequence 215 aagacattaa aatattgatt g 21 21621 DNA Artificial Sequence target sequence 216 aaaatattga ttgcatctcc a21 217 21 DNA Artificial Sequence target sequence 217 aaatattgattgcatctcca t 21 218 21 DNA Artificial Sequence target sequence 218aatattgatt gcatctccat c 21 219 21 DNA Artificial Sequence targetsequence 219 aaagaaacta ctagtgccac a 21 220 21 DNA Artificial Sequencetarget sequence 220 aagaaactac tagtgccaca t 21 221 21 DNA ArtificialSequence target sequence 221 aaactactag tgccacatca t 21 222 21 DNAArtificial Sequence target sequence 222 aactactagt gccacatcat c 21 22321 DNA Artificial Sequence target sequence 223 aaagtcggac agcctcacca a21 224 21 DNA Artificial Sequence target sequence 224 aagtcggacagcctcaccaa a 21 225 21 DNA Artificial Sequence target sequence 225aaacagagca ggaaaaggag t 21 226 21 DNA Artificial Sequence targetsequence 226 aacagagcag gaaaaggagt c 21 227 21 DNA Artificial Sequencetarget sequence 227 aaaaggagtc atagaacaga c 21 228 21 DNA ArtificialSequence target sequence 228 aaaggagtca tagaacagac a 21 229 21 DNAArtificial Sequence target sequence 229 aaggagtcat agaacagaca g 21 23021 DNA Artificial Sequence target sequence 230 aacagacaga aaaatctcat c21 231 21 DNA Artificial Sequence target sequence 231 aaaaatctcatccaagaagc c 21 232 21 DNA Artificial Sequence target sequence 232aaaatctcat ccaagaagcc c 21 233 21 DNA Artificial Sequence targetsequence 233 aaatctcatc caagaagccc t 21 234 21 DNA Artificial Sequencetarget sequence 234 aatctcatcc aagaagccct a 21 235 21 DNA ArtificialSequence target sequence 235 aagaagccct aacgtgttat c 21 236 21 DNAArtificial Sequence target sequence 236 aagccctaac gtgttatctg t 21 23721 DNA Artificial Sequence target sequence 237 aacgtgttat ctgtcgcttt g21 238 21 DNA Artificial Sequence target sequence 238 aaagaactacagttcctgag g 21 239 21 DNA Artificial Sequence target sequence 239aagaactaca gttcctgagg a 21 240 21 DNA Artificial Sequence targetsequence 240 aactacagtt cctgaggaag a 21 241 21 DNA Artificial Sequencetarget sequence 241 aagaactaaa tccaaagata c 21 242 21 DNA ArtificialSequence target sequence 242 aactaaatcc aaagatacta g 21 243 21 DNAArtificial Sequence target sequence 243 aaatccaaag atactagctt t 21 24421 DNA Artificial Sequence target sequence 244 aatccaaaga tactagcttt g21 245 21 DNA Artificial Sequence target sequence 245 aaagatactagctttgcaga a 21 246 21 DNA Artificial Sequence target sequence 246aagatactag ctttgcagaa t 21 247 21 DNA Artificial Sequence targetsequence 247 aatgctcaga gaaagcgaaa a 21 248 21 DNA Artificial Sequencetarget sequence 248 aaagcgaaaa atggaacatg a 21 249 21 DNA ArtificialSequence target sequence 249 aagcgaaaaa tggaacatga t 21 250 21 DNAArtificial Sequence target sequence 250 aaaaatggaa catgatggtt c 21 25121 DNA Artificial Sequence target sequence 251 aaaatggaac atgatggttc a21 252 21 DNA Artificial Sequence target sequence 252 aaatggaacatgatggttca c 21 253 21 DNA Artificial Sequence target sequence 253aatggaacat gatggttcac t 21 254 21 DNA Artificial Sequence targetsequence 254 aacatgatgg ttcacttttt c 21 255 21 DNA Artificial Sequencetarget sequence 255 aagcagtagg aattggaaca t 21 256 21 DNA ArtificialSequence target sequence 256 aattggaaca ttattacagc a 21 257 21 DNAArtificial Sequence target sequence 257 aacattatta cagcagccag a 21 25821 DNA Artificial Sequence target sequence 258 aaacgtgtaa aaggatgcaa a21 259 21 DNA Artificial Sequence target sequence 259 aacgtgtaaaaggatgcaaa t 21 260 21 DNA Artificial Sequence target sequence 260aaaaggatgc aaatctagtg a 21 261 21 DNA Artificial Sequence targetsequence 261 aaaggatgca aatctagtga a 21 262 21 DNA Artificial Sequencetarget sequence 262 aaggatgcaa atctagtgaa c 21 263 21 DNA ArtificialSequence target sequence 263 aaatctagtg aacagaatgg a 21 264 21 DNAArtificial Sequence target sequence 264 aatctagtga acagaatgga a 21 26521 DNA Artificial Sequence target sequence 265 aacagaatgg aatggagcaa a21 266 21 DNA Artificial Sequence target sequence 266 aatggaatggagcaaaagac a 21 267 21 DNA Artificial Sequence target sequence 267aatggagcaa aagacaatta t 21 268 21 DNA Artificial Sequence targetsequence 268 aaaagacaat tattttaata c 21 269 21 DNA Artificial Sequencetarget sequence 269 aaagacaatt attttaatac c 21 270 21 DNA ArtificialSequence target sequence 270 aagacaatta ttttaatacc c 21 271 21 DNAArtificial Sequence target sequence 271 aattatttta ataccctctg a 21 27221 DNA Artificial Sequence target sequence 272 aataccctct gatttagcat g21 273 21 DNA Artificial Sequence target sequence 273 aatcaatggatgaaagtgga t 21 274 21 DNA Artificial Sequence target sequence 274aatggatgaa agtggattac c 21 275 21 DNA Artificial Sequence targetsequence 275 aaagtggatt accacagctg a 21 276 21 DNA Artificial Sequencetarget sequence 276 aagtggatta ccacagctga c 21 277 21 DNA ArtificialSequence target sequence 277 catcagttgc cacttccaca t 21 278 21 DNAArtificial Sequence target sequence 278 cttggatggt tttgttatgg t 21 27921 DNA Artificial Sequence target sequence 279 atgggattaa ctcagtttga a21 280 21 DNA Artificial Sequence target sequence 280 gtctgcaacatggaaggtat t 21 281 21 DNA Artificial Sequence target sequence 281cattcctcac ccatcaaata t 21 282 21 DNA Artificial Sequence targetsequence 282 aggccgctca atttatgaat a 21 283 21 DNA Artificial Sequencetarget sequence 283 tcatatataa caccaagaat t 21 284 21 DNA ArtificialSequence target sequence 284 tgtccttaaa ccggttgaat c 21 285 21 DNAArtificial Sequence target sequence 285 agcctctttg acaaacttaa g 21 28621 DNA Artificial Sequence target sequence 286 atgaccagca acttgaggaa g21 287 21 DNA Artificial Sequence target sequence 287 cattacccaccgctgaaacg c 21 288 21 DNA Artificial Sequence target sequence 288agattcagga tcagacacct a 21 289 21 DNA Artificial Sequence targetsequence 289 atagtgatat ggtcaatgaa t 21 290 21 DNA Artificial Sequencetarget sequence 290 acacagattt agacttggag a 21 291 21 DNA ArtificialSequence target sequence 291 cacagttaca gtattccagc a 21 292 21 DNAArtificial Sequence target sequence 292 attgattgca tctccatctc c 21 29321 DNA Artificial Sequence target sequence 293 atactagctt tgcagaatgc t21 294 21 DNA Artificial Sequence target sequence 294 attattacagcagccagacg a 21 295 21 DNA Artificial Sequence target sequence 295acaattattt taataccctc t 21 296 21 DNA Artificial Sequence targetsequence 296 accagttatg attgtgaagt t 21 297 21 DNA Artificial Sequencetarget sequence 297 aactaactgg acacagtgtg t 21 298 21 DNA ArtificialSequence siRNA sense strand 298 cuaacuggac acagugugut t 21 299 21 DNAArtificial Sequence siRNA antisense strand 299 acacacugug uccaguuagt t21

We claim:
 1. An isolated siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human HIF-1 alpha mRNA,or an alternative splice form, mutant or cognate thereof.
 2. The siRNAof claim 1, wherein the human HIF-1 alpha mRNA is SEQ ID NO:
 1. 3. ThesiRNA of claim 1, wherein the cognate of the human HIF-1 alpha mRNAsequence is rat HIF-1 alpha mRNA or mouse HIF-1 alpha mRNA.
 4. The siRNAof claim 1, wherein the sense RNA strand comprises one RNA molecule, andthe antisense RNA strand comprises one RNA molecule.
 5. The siRNA ofclaim 1, wherein the sense and antisense RNA strands forming the RNAduplex are covalently linked by a single-stranded hairpin.
 6. The siRNAof claim 1, wherein the siRNA further comprises non-nucleotide material.7. The siRNA of claim 1, wherein the siRNA further comprises anaddition, deletion, substitution or alteration of one or morenucleotides.
 8. The siRNA of claim 1, wherein the sense and antisenseRNA strands are stabilized against nuclease degradation.
 9. The siRNA ofclaim 1, further comprising a 3′ overhang.
 10. The siRNA of claim 9,wherein the 3′ overhang comprises from 1 to about 6 nucleotides.
 11. ThesiRNA of claim 9, wherein the 3′ overhang comprises about 2 nucleotides.12. The siRNA of claim 5, wherein the sense RNA strand comprises a first3′ overhang, and the antisense RNA strand comprises a second 3′overhang.
 13. The siRNA of claim 12, wherein the first and second 3′overhangs separately comprise from 1 to about 6 nucleotides.
 14. ThesiRNA of claim 13, wherein the first 3′ overhang comprises adinucleotide and the second 3′ overhang comprises a dinucleotide. 15.The siRNA of claim 14, where the dinucleotide comprising the first andsecond 3′ overhangs is dithymidylic acid (TT) or diuridylic acid (uu).16. The siRNA of claim 9, wherein the 3′ overhang is stabilized againstnuclease degradation.
 17. A retinal pigment epithelial cell comprisingthe siRNA of claim
 1. 18. A recombinant plasmid comprising nucleic acidsequences for expressing an siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human HIF-1 alpha mRNA,or an alternative splice form, mutant or cognate thereof.
 19. Therecombinant plasmid of claim 18, wherein the nucleic acid sequences forexpressing the siRNA comprise an inducible or regulatable promoter. 20.The recombinant plasmid of claim 18, wherein the nucleic acid sequencesfor expressing the siRNA comprise a sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and an antisense RNA strand coding sequencein operable connection with a polyT termination sequence under thecontrol of a human U6 RNA promoter.
 21. The recombinant plasmid of claim20, wherein the plasmid is pAAVsiRNA.
 22. A recombinant viral vectorcomprising nucleic acid sequences for expressing an siRNA comprising asense RNA strand and an antisense RNA strand, wherein the sense and anantisense RNA strands form an RNA duplex, and wherein the sense RNAstrand comprises a nucleotide sequence substantially identical to atarget sequence of about 19 to about 25 contiguous nucleotides in humanHIF-1 alpha mRNA, or an alternative splice form, mutant or cognatethereof.
 23. The recombinant viral vector of claim 22, wherein thenucleic acid sequences for expressing the siRNA comprise an inducible orregulatable promoter.
 24. The recombinant viral vector of claim 22,wherein the nucleic acid sequences for expressing the siRNA comprise asense RNA strand coding sequence in operable connection with a polyTtermination sequence under the control of a human U6 RNA promoter, andan antisense RNA strand coding sequence in operable connection with apolyT termination sequence under the control of a human U6 RNA promoter.25. The recombinant viral vector of claim 22, wherein the recombinantviral vector is selected from the group consisting of an adenoviralvector, an adeno-associated viral vector, a lentiviral vector, aretroviral vector, and a herpes virus vector.
 26. The recombinant viralvector of claim 22, wherein the recombinant viral vector is pseudotypedwith surface proteins from vesicular stomatitis virus, rabies virus,Ebola virus, or Mokola virus.
 27. The recombinant viral vector of claim25, wherein the recombinant viral vector comprises an adeno-associatedviral vector.
 28. A pharmaceutical composition comprising an siRNA and apharmaceutically acceptable carrier, wherein the siRNA comprises a senseRNA strand and an antisense RNA strand, wherein the sense and anantisense RNA strands form an RNA duplex, and wherein the sense RNAstrand comprises a nucleotide sequence substantially identical to atarget sequence of about 19 to about 25 contiguous nucleotides in humanHIF-1 alpha mRNA, or an alternative splice form, mutant or cognatethereof.
 29. The pharmaceutical composition of claim 28, furthercomprising lipofectin, lipofectamine, cellfectin, polycations, orliposomes.
 30. A pharmaceutical composition comprising the plasmid ofclaim 18, or a physiologically acceptable salt thereof, and apharmaceutically acceptable carrier.
 31. The pharmaceutical compositionof claim 30, further comprising lipofectin, lipofectamine, cellfectin,polycations, or liposomes.
 32. A pharmaceutical composition comprisingthe viral vector of claim 22 and a pharmaceutically acceptable carrier.33. A method of inhibiting expression of human HIF-1 alpha mRNA, or analternative splice form, mutant or cognate thereof, comprisingadministering to a subject an effective amount of an siRNA comprising asense RNA strand and an antisense RNA strand, wherein the sense and anantisense RNA strands form an RNA duplex, and wherein the sense RNAstrand comprises a nucleotide sequence substantially identical to atarget sequence of about 19 to about 25 contiguous nucleotides in humanHIF-1 alpha mRNA, or an alternative splice form, mutant or cognatethereof, such that human HIF-1 alpha mRNA, or an alternative spliceform, mutant or cognate thereof, is degraded.
 34. The method of claim33, wherein the subject is a human being.
 35. The method of claim 33,wherein expression of human HIF-1 alpha mRNA, or an alternative spliceform, mutant or cognate thereof is inhibited in one or both eyes of thesubject.
 36. The method of claim 33, wherein expression of human HIF-1alpha mRNA, or an alternative splice form, mutant or cognate thereof isinhibited in retinal pigment epithelial cells of the subject.
 37. Themethod of claim 33, wherein the effective amount of the siRNA is anamount which provides an intercellular concentration at or near theneovascularization site of from about 1 nM to about 100 nM.
 38. Themethod of claim 33, wherein the siRNA is administered in conjunctionwith a delivery reagent.
 39. The method of claim 38, wherein thedelivery agent is selected from the group consisting of lipofectin,lipofectamine, cellfectin, polycations, and liposomes.
 40. The method ofclaim 39, wherein the delivery agent is a liposome.
 41. The method claim40, wherein the liposome comprises a ligand which targets the liposometo cells at or near the site of angiogenesis.
 42. The method of claim41, wherein the ligand binds to receptors on tumor cells or vascularendothelial cells.
 43. The method of claim 42, wherein the ligandcomprises a monoclonal antibody.
 44. The method of claim 40, wherein theliposome is modified with an opsonization-inhibition moiety.
 45. Themethod of claim 44, wherein the opsonization-inhibiting moiety comprisesa PEG, PPG, or derivatives thereof.
 46. The method of claim 33, whereinthe siRNA is expressed from a recombinant plasmid
 47. The method ofclaim 33, wherein the siRNA is expressed from a recombinant viralvector.
 48. The method of claim 47, wherein the recombinant viral vectorcomprises an adenoviral vector, an adeno-associated viral vector, alentiviral vector, a retroviral vector, or a herpes virus vector. 49.The method of claim 48, wherein the recombinant viral vector ispseudotyped with surface proteins from vesicular stomatitis virus,rabies virus, Ebola virus, or Mokola virus.
 50. The method of claim 47,wherein the recombinant viral vector comprises an adeno-associated viralvector.
 51. The method of claim 33, wherein the siRNA is administered byan enteral administration route.
 52. The method of claim 51, wherein theenteral administration route is selected from the group consisting oforal, rectal, and intranasal.
 53. The method of claim 33, wherein thesiRNA is administered by a parenteral administration route.
 54. Themethod of claim 53, wherein the parenteral administration route isselected from the group consisting of intravascular administration,peri- and intra-tissue administration, subcutaneous injection ordeposition, subcutaneous infusion, intraocular administration, anddirect application at or near the site of neovascularization.
 55. Themethod of claim 54, wherein the intravascular administration is selectedfrom the group consisting of intravenous bolus injection, intravenousinfusion, intra-arterial bolus injection, intra-arterial infusion andcatheter instillation into the vasculature.
 56. The method of claim 54,wherein the peri- and intra-tissue injection comprises peri-tumoralinjection or intra-tumoral injection.
 57. The method of claim 54,wherein the intraocular administration comprises intravitreal,intraretinal, subretinal, subtenon, peri- and retro-orbital,trans-corneal or trans-scleral administration.
 58. The method of claim54, wherein the direct application at or near the site ofneovascularization comprises application by catheter, corneal pellet,eye dropper, suppository, an implant comprising a porous material, animplant comprising a non-porous material, or an implant comprising agelatinous material.
 59. The method of claim 54, wherein the site ofneovascularization is in the eye, and the direct application at or nearthe site of neovascularization comprises application by an ocularimplant.
 60. The method of claim 59, wherein the ocular implant isbiodegradable.
 61. A method of inhibiting angiogenesis in a subject,comprising administering to a subject an effective amount of an siRNAcomprising a sense RNA strand and an antisense RNA strand, wherein thesense and an antisense RNA strands form an RNA duplex, and wherein thesense RNA strand comprises a nucleotide sequence substantially identicalto a target sequence of about 19 to about 25 contiguous nucleotides inhuman HIF-1 alpha mRNA, or an alternative splice form, mutant or cognatethereof.
 62. The method of claim 61, wherein the angiogenesis ispathogenic.
 63. The method of claim 61, wherein the angiogenesis isnon-pathogenic.
 64. The method of claim 63, wherein the non-pathogenicangiogenesis is associated with production of fatty tissues orcholesterol production.
 65. The method of claim 63, wherein thenon-pathogenic angiogenesis comprises endometrial neovascularization.66. The method of claim 61, wherein the angiogenesis is inhibited in oneor both eyes of the subject.
 67. A method of treating an angiogenicdisease in a subject, comprising administering to a subject an effectiveamount of an siRNA comprising a sense RNA strand and an antisense RNAstrand, wherein the sense and an antisense RNA strands form an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequencesubstantially identical to a target sequence of about 19 to about 25contiguous nucleotides in human HIF-1 alpha mRNA, or an alternativesplice form, mutant or cognate thereof, such that angiogenesisassociated with the angiogenic disease is inhibited.
 68. The method ofclaim 67, wherein the angiogenic disease comprises a tumor associatedwith a cancer.
 69. The method of claim 68, wherein the cancer isselected from the group consisting of breast cancer, lung cancer, headand neck cancer, brain cancer, abdominal cancer, colon cancer,colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma,liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovariancancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm'stumor, multiple myeloma, skin cancer, lymphoma, and blood cancer. 70.The method of claim 67, wherein the angiogenic disease is selected fromthe group consisting of diabetic retinopathy, age-related maculardegeneration, and inflammatory diseases.
 71. The method of claim 70,wherein the inflammatory disease is psoriasis or rheumatoid arthritis.72. The method of claim 70, wherein the angiogenic disease isage-related macular degeneration.
 73. The method of claim 67, whereinthe siRNA is administered in combination with a pharmaceutical agent fortreating the angiogenic disease, which pharmaceutical agent is differentfrom the siRNA.
 74. The method of claim 73, wherein the angiogenicdisease is cancer, and the pharmaceutical agent comprises achemotherapeutic agent.
 75. The method of claim 73, wherein thechemotherapeutic agent is selected from the group consisting ofcisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin,daunorubicin, and tamoxifen.
 76. The method of claim 67, wherein thesiRNA is administered to a subject in combination with anothertherapeutic method designed to treat the angiogenic disease.
 77. Themethod of claim 76, wherein the angiogenic disease is cancer, and thesiRNA is administered in combination with radiation therapy,chemotherapy or surgery.